Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease
TL;DR: A protocol that detects neutral lipids and lipid droplet (LD) morphology by oil red O (ORO) staining of sections from frozen tissues is presented, allowing for easy estimation of tissue lipid content and distribution using only basic laboratory and computer equipment.
Abstract: Excess lipid accumulation in peripheral tissues is a key feature of many metabolic diseases. Therefore, techniques for imaging and quantifying lipids in various tissues are important for understanding and evaluating the overall metabolic status of a research subject. Here we present a protocol that detects neutral lipids and lipid droplet (LD) morphology by oil red O (ORO) staining of sections from frozen tissues. The method allows for easy estimation of tissue lipid content and distribution using only basic laboratory and computer equipment. Furthermore, the procedure described here is well suited for the comparison of different metabolically challenged animal models. As an example, we include data on muscular and hepatic lipid accumulation in diet-induced and genetically induced diabetic mice. The experimental description presents details for optimal staining of lipids using ORO, including tissue collection, sectioning, staining, imaging and measurements of tissue lipids, in a time frame of less than 2 d.
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TL;DR: It is demonstrated that cellular senescence drives hepatic Steatosis and elimination of senescent cells may be a novel therapeutic strategy to reduce steatosis.
Abstract: The incidence of non-alcoholic fatty liver disease (NAFLD) increases with age. Cellular senescence refers to a state of irreversible cell-cycle arrest combined with the secretion of proinflammatory cytokines and mitochondrial dysfunction. Senescent cells contribute to age-related tissue degeneration. Here we show that the accumulation of senescent cells promotes hepatic fat accumulation and steatosis. We report a close correlation between hepatic fat accumulation and markers of hepatocyte senescence. The elimination of senescent cells by suicide gene-meditated ablation of p16Ink4a-expressing senescent cells in INK-ATTAC mice or by treatment with a combination of the senolytic drugs dasatinib and quercetin (D+Q) reduces overall hepatic steatosis. Conversely, inducing hepatocyte senescence promotes fat accumulation in vitro and in vivo. Mechanistically, we show that mitochondria in senescent cells lose the ability to metabolize fatty acids efficiently. Our study demonstrates that cellular senescence drives hepatic steatosis and elimination of senescent cells may be a novel therapeutic strategy to reduce steatosis.
603 citations
TL;DR: A diet-induced animal model of non-alcoholic fatty liver disease (DIAMOND) that recapitulates the key physiological, metabolic, histologic, transcriptomic and cell-signaling changes seen in humans with progressive NASH is described.
345 citations
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...The presence of steatosis was further confirmed used Oil-RedO stains in frozen sections using standard methods [8]....
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Journal Article•
TL;DR: In this article, the authors proposed VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipidtransport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.
Abstract: The prevalence of type 2 diabetes is rapidly increasing, with severe socioeconomic impacts. Excess lipid deposition in peripheral tissues impairs insulin sensitivity and glucose uptake, and has been proposed to contribute to the pathology of type 2 diabetes. However, few treatment options exist that directly target ectopic lipid accumulation. Recently it was found that vascular endothelial growth factor B (VEGF-B) controls endothelial uptake and transport of fatty acids in heart and skeletal muscle. Here we show that decreased VEGF-B signalling in rodent models of type 2 diabetes restores insulin sensitivity and improves glucose tolerance. Genetic deletion of Vegfb in diabetic db/db mice prevented ectopic lipid deposition, increased muscle glucose uptake and maintained normoglycaemia. Pharmacological inhibition of VEGF-B signalling by antibody administration to db/db mice enhanced glucose tolerance, preserved pancreatic islet architecture, improved β-cell function and ameliorated dyslipidaemia, key elements of type 2 diabetes and the metabolic syndrome. The potential use of VEGF-B neutralization in type 2 diabetes was further elucidated in rats fed a high-fat diet, in which it normalized insulin sensitivity and increased glucose uptake in skeletal muscle and heart. Our results demonstrate that the vascular endothelium can function as an efficient barrier to excess muscle lipid uptake even under conditions of severe obesity and type 2 diabetes, and that this barrier can be maintained by inhibition of VEGF-B signalling. We propose VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipid-transport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.
225 citations
TL;DR: The findings uncover a prometastatic lipogenic program and lend direct genetic and experimental support to the notion that a Western HFD can promote metastasis and show that a high-fat diet induces lipid accumulation in prostate tumors and is sufficient to drive metastasis.
Abstract: Lipids, either endogenously synthesized or exogenous, have been linked to human cancer. Here we found that PML is frequently co-deleted with PTEN in metastatic human prostate cancer (CaP). We demonstrated that conditional inactivation of Pml in the mouse prostate morphs indolent Pten-null tumors into lethal metastatic disease. We identified MAPK reactivation, subsequent hyperactivation of an aberrant SREBP prometastatic lipogenic program, and a distinctive lipidomic profile as key characteristic features of metastatic Pml and Pten double-null CaP. Furthermore, targeting SREBP in vivo by fatostatin blocked both tumor growth and distant metastasis. Importantly, a high-fat diet (HFD) induced lipid accumulation in prostate tumors and was sufficient to drive metastasis in a nonmetastatic Pten-null mouse model of CaP, and an SREBP signature was highly enriched in metastatic human CaP. Thus, our findings uncover a prometastatic lipogenic program and lend direct genetic and experimental support to the notion that a Western HFD can promote metastasis.
207 citations
TL;DR: In this paper, the mitochondrial uncoupling protein-2 (UCP2) was shown to regulate caspase-1 activation through the stimulation of lipid synthesis in macrophages.
Abstract: Cellular lipid metabolism has been linked to immune responses; however, the precise mechanisms by which de novo fatty acid synthesis can regulate inflammatory responses remain unclear. The NLRP3 inflammasome serves as a platform for caspase-1–dependent maturation and secretion of proinflammatory cytokines. Here, we demonstrated that the mitochondrial uncoupling protein-2 (UCP2) regulates NLRP3-mediated caspase-1 activation through the stimulation of lipid synthesis in macrophages. UCP2-deficient mice displayed improved survival in a mouse model of polymicrobial sepsis. Moreover, UCP2 expression was increased in human sepsis. Consistently, UCP2-deficient mice displayed impaired lipid synthesis and decreased production of IL-1β and IL-18 in response to LPS challenge. In macrophages, UCP2 deficiency suppressed NLRP3-mediated caspase-1 activation and NLRP3 expression associated with inhibition of lipid synthesis. In UCP2-deficient macrophages, inhibition of lipid synthesis resulted from the downregulation of fatty acid synthase (FASN), a key regulator of fatty acid synthesis. FASN inhibition by shRNA and treatment with the chemical inhibitors C75 and cerulenin suppressed NLRP3-mediated caspase-1 activation and inhibited NLRP3 and pro–IL-1β gene expression in macrophages. In conclusion, our results suggest that UCP2 regulates the NLRP3 inflammasome by inducing the lipid synthesis pathway in macrophages. These results identify UCP2 as a potential therapeutic target in inflammatory diseases such as sepsis.
204 citations
References
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TL;DR: The origins, challenges and solutions of NIH Image and ImageJ software are discussed, and how their history can serve to advise and inform other software projects.
Abstract: For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
44,587 citations
TL;DR: The hypothesis that an increase in plasma fatty acid concentration results in a increase in intracellular fatty acyl-CoA and DAG concentrations, which results in activation of PKC-θ leading to increased IRS-1 Ser307 phosphorylation is supported.
1,562 citations
TL;DR: This unifying hypothesis accounts for the mechanism of insulin resistance in obesity, type 2 diabetes, lipodystrophy, and ageing; and the insulin-sensitising effects of thiazolidinediones.
1,000 citations
TL;DR: A fast and simple method to quantitate the extent of adipose conversion by staining the accumulated lipid with Oil red O and determining the amount of extracted dye at 510 nm is reported.
Abstract: Cultured 3T3-F442A cells differentiate into adipocytes and accumulate lipid droplets in the cytoplasm. When fat cells are stained with Oil red O, the degree of staining seems to be proportional to the extent of cell differentiation. We report here a fast and simple method to quantitate the extent of adipose conversion by staining the accumulated lipid with Oil red O and determining the amount of extracted dye at 510 nm. The results show that Oil red O specifically stains triglycerides and cholesteryl oleate but no other lipids. This technique is a valuable tool for processing large numbers of cell cultures or samples in which adipose differentiation and/or accumulated triglycerides is to be quantitated.
935 citations
TL;DR: Quiescent stellate cells presumably contribute to the control of blood flow through the sinusoidal capillaries and are important sources of paracrine, autocrine, juxtACrine, and chemoattractant factors that maintain homeostasis in the microenvironment of the hepatic sinusoid.
Abstract: In 1876, von Kupffer described liver Sternzellen (star-shaped cells). The functions of these cells remained enigmatic for 75 years until Ito observed lipid-containing perisinusoidal cells in human liver. In 1971, Wake demonstrated that the Sternzellen of von Kupffer and the fat-storing cells described by Ito were identical. Wake also established that these cells were important sites of vitamin A storage. Soon thereafter, Kent and Popper demonstrated that the stellate cells were intimately linked to the pathogenesis of hepatic fibrosis. Since then, these cells have been studied in detail. Quiescent stellate cells represent 5-8% of the total number of liver cells. They play a cardinal role in storage and controlled release of retinoids. They control extracellular matrix (ECM) turnover in the space of Disse by secreting the correct amounts of a limited number of ECM molecules, and by releasing matrix metalloproteinases and their inhibitors. By virtue of their long cytoplasmic processes, quiescent stellate cells presumably contribute to the control of blood flow through the sinusoidal capillaries. They are important sources of paracrine, autocrine, juxtacrine, and chemoattractant factors that maintain homeostasis in the microenvironment of the hepatic sinusoid.
701 citations