Publications by authors named "Dagmar Kratky"

85 Publications

Loss of function of lysosomal acid lipase (LAL) profoundly impacts osteoblastogenesis and increases fracture risk in humans.

Bone 2021 Jul 7;148:115946. Epub 2021 Apr 7.

Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME 04074, USA; Center for Bone Biology, Division of Clinical Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA; Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Electronic address:

Lysosomal acid lipase (LAL) is essential for cholesteryl ester (CE) and triacylglycerol (TAG) hydrolysis in the lysosome. Clinically, an autosomal recessive LIPA mutation causes LAL deficiency (LALD), previously described as Wolman Disease or Cholesteryl Ester Storage Disease (CESD). LAL-D is associated with ectopic lipid accumulation in the liver, small intestine, spleen, adrenal glands, and blood. Considering the importance of unesterified cholesterol and fatty acids in bone metabolism, we hypothesized that LAL is essential for bone formation, and ultimately, skeletal health. To investigate the role of LAL in skeletal homeostasis, we used LAL-deficient () mice, in vitro osteoblast cultures, and novel clinical data from LAL-D patients. Both male and female LAL mice demonstarted lower trabecular and cortical bone parameters , which translated to reduced biomechanical properties. Further histological analyses revealed that LAL mice had fewer osteoblasts, with no change in osteoclast or marrow adipocyte numbers. In studying the cell-autonomous role of LAL, we observed impaired differentiation of LAL calvarial osteoblasts and in bone marrow stromal cells treated with the LAL inhibitor lalistat. Consistent with LAL's role in other tissues, lalistat resulted in profound lipid puncta accumulation and an altered intracellular lipid profile. Finally, we analyzed a large de-identified national insurance database (i.e. 2016/2017 Optum Clinformatics®) which revealed that adults (≥18 years) with CESD (n = 3076) had a higher odds ratio (OR = 1.21; 95% CI = 1.03-1.41) of all-cause fracture at any location compared to adults without CESD (n = 13.7 M) after adjusting for demographic variables and osteoporosis. These data demonstrate that alterations in LAL have significant clinical implications related to fracture risk and that LAL's modulation of lipid metabolism is a critical for osteoblast function.
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http://dx.doi.org/10.1016/j.bone.2021.115946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8108562PMC
July 2021

Tissue-Specific Landscape of Metabolic Dysregulation during Ageing.

Biomolecules 2021 02 7;11(2). Epub 2021 Feb 7.

Gottfried Schatz Research Center for Cell Signaling, Metabolism and Ageing, Molecular Biology and Biochemistry, Medical University of Graz, 8010 Graz, Austria.

The dysregulation of cellular metabolism is a hallmark of ageing. To understand the metabolic changes that occur as a consequence of the ageing process and to find biomarkers for age-related diseases, we conducted metabolomic analyses of the brain, heart, kidney, liver, lung and spleen in young (9-10 weeks) and old (96-104 weeks) wild-type mice [mixed genetic background of 129/J and C57BL/6] using NMR spectroscopy. We found differences in the metabolic fingerprints of all tissues and distinguished several metabolites to be altered in most tissues, suggesting that they may be universal biomarkers of ageing. In addition, we found distinct tissue-clustered sets of metabolites throughout the organism. The associated metabolic changes may reveal novel therapeutic targets for the treatment of ageing and age-related diseases. Moreover, the identified metabolite biomarkers could provide a sensitive molecular read-out to determine the age of biologic tissues and organs and to validate the effectiveness and potential off-target effects of senolytic drug candidates on both a systemic and tissue-specific level.
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http://dx.doi.org/10.3390/biom11020235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7914945PMC
February 2021

ATG7 is dispensable for LC3-PE conjugation in thioglycolate-elicited mouse peritoneal macrophages.

Autophagy 2021 Jan 18:1-6. Epub 2021 Jan 18.

Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.

Thioglycolate-elicited macrophages exhibit abundant conjugation of LC3 with PE (LC3-II). Among other autophagy-related (ATG) proteins, it is proposed that, like in yeast, both ATG5 and ATG7 are essential for LC3 conjugation. Using -deficient () and macrophages, we provide evidence that loss of ATG5 but not of ATG7 resulted in LC3-II depletion. Accumulation of LC3-II in elicited macrophages in response to bafilomycin A validated these data. Furthermore, complete loss of ATG3 in macrophages demonstrated that ATG7 and ATG3 are dispensable for LC3-PE conjugation. In contrast to thioglycolate-elicited macrophages, naïve peritoneal and bone marrow-derived macrophages exhibited no LC3-II, even under inflammatory stimuli . Hence, the macrophage metabolic status dictates the level of LC3-PE conjugation with a supportive but nonessential role of ATG7, disclosing the eukaryotic exception from the LC3 lipidation model based on yeast data. : ATG: autophagy-related; BM: bone marrow; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PE: phosphatidylethanolamine.
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http://dx.doi.org/10.1080/15548627.2021.1874132DOI Listing
January 2021

Endothelial Lipase Modulates Paraoxonase 1 Content and Arylesterase Activity of HDL.

Int J Mol Sci 2021 Jan 13;22(2). Epub 2021 Jan 13.

Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria.

Endothelial lipase (EL) is a strong modulator of the high-density lipoprotein (HDL) structure, composition, and function. Here, we examined the impact of EL on HDL paraoxonase 1 (PON1) content and arylesterase (AE) activity in vitro and in vivo. The incubation of HDL with EL-overexpressing HepG2 cells decreased HDL size, PON1 content, and AE activity. The EL modification of HDL did not diminish the capacity of HDL to associate with PON1 when EL-modified HDL was incubated with PON1-overexpressing cells. The overexpression of EL in mice significantly decreased HDL serum levels but unexpectedly increased HDL PON1 content and HDL AE activity. Enzymatically inactive EL had no effect on the PON1 content of HDL in mice. In healthy subjects, EL serum levels were not significantly correlated with HDL levels. However, HDL PON1 content was positively associated with EL serum levels. The EL-induced changes in the HDL-lipid composition were not linked to the HDL PON1 content. We conclude that primarily, the interaction of enzymatically active EL with HDL, rather than EL-induced alterations in HDL size and composition, causes PON1 displacement from HDL in vitro. In vivo, the EL-mediated reduction of HDL serum levels and the consequently increased PON1-to-HDL ratio in serum increase HDL PON1 content and AE activity in mice. In humans, additional mechanisms appear to underlie the association of EL serum levels and HDL PON1 content.
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http://dx.doi.org/10.3390/ijms22020719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828365PMC
January 2021

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy.

Histochem Cell Biol 2021 Jan 6. Epub 2021 Jan 6.

Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Division of Cell Biology, Histology and Embryology, Medical University of Graz, Neue Stiftingtalstraße 6/II, 8010, Graz, Austria.

Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. "biobanking" of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.
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http://dx.doi.org/10.1007/s00418-020-01952-zDOI Listing
January 2021

Hepatic synthesis of triacylglycerols containing medium-chain fatty acids is dominated by diacylglycerol acyltransferase 1 and efficiently inhibited by etomoxir.

Mol Metab 2021 03 23;45:101150. Epub 2020 Dec 23.

LIMES Life and Medical Sciences Institute, Rheinische Friedrich-Wilhelms-Universität Bonn, Carl-Troll-Str. 31, 53115 Bonn, Germany. Electronic address:

Objective: Medium-chain fatty acids (MCFAs) play an increasing role in human nutrition. In the liver, one fraction is used for synthesis of MCFA-containing triacylglycerol (MCFA-TG), and the rest is used for oxidative energy production or ketogenesis. We investigated which enzymes catalyse the synthesis of MCFA-TG and how inhibition of MCFA-TG synthesis or fatty acid (FA) oxidation influences the metabolic fate of the MCFAs.

Methods: FA metabolism was followed by time-resolved tracing of alkyne-labelled FAs in freshly isolated mouse hepatocytes. Quantitative data were obtained by mass spectrometry of several hundred labelled lipid species. Wild-type hepatocytes and cells from diacylglycerol acyltransferase (DGAT)1 mice were treated with inhibitors against DGAT1, DGAT2, or FA β-oxidation.

Results: Inhibition or deletion of DGAT1 resulted in a reduction of MCFA-TG synthesis by 70%, while long-chain (LC)FA-TG synthesis was reduced by 20%. In contrast, DGAT2 inhibition increased MCFA-TG formation by 50%, while LCFA-TG synthesis was reduced by 5-25%. Inhibition of β-oxidation by the specific inhibitor teglicar strongly increased MCFA-TG synthesis. In contrast, the widely used β-oxidation inhibitor etomoxir blocked MCFA-TG synthesis, phenocopying DGAT1 inhibition.

Conclusions: DGAT1 is the major enzyme for hepatic MCFA-TG synthesis. Its loss can only partially be compensated by DGAT2. Specific inhibition of β-oxidation leads to a compensatory increase in MCFA-TG synthesis, whereas etomoxir blocks both β-oxidation and MCFA-TG synthesis, indicating a strong off-target effect on DGAT1.
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http://dx.doi.org/10.1016/j.molmet.2020.101150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843514PMC
March 2021

Spatially Resolved Activity-based Proteomic Profiles of the Murine Small Intestinal Lipases.

Mol Cell Proteomics 2020 12 6;19(12):2104-2115. Epub 2020 Oct 6.

Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria; Diagnostic and Research Institute of Pathology, Medical University Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Despite the crucial function of the small intestine in nutrient uptake our understanding of the molecular events underlying the digestive function is still rudimentary. Recent studies demonstrated that enterocytes do not direct the entire dietary triacylglycerol toward immediate chylomicron synthesis. Especially after high-fat challenges, parts of the resynthesized triacylglycerol are packaged into cytosolic lipid droplets for transient storage in the endothelial layer of the small intestine. The reason for this temporary storage of triacylglycerol is not completely understood. To utilize lipids from cytosolic lipid droplets for chylomicron synthesis in the endoplasmic reticulum, stored triacylglycerol has to be hydrolyzed either by cytosolic lipolysis or lipophagy. Interestingly, triacylglycerol storage and chylomicron secretion rates are unevenly distributed along the small intestine, with the proximal jejunum exhibiting the highest intermittent storage capacity. We hypothesize that correlating hydrolytic enzyme activities with the reported distribution of triacylglycerol storage and chylomicron secretion in different sections of the small intestine is a promising strategy to determine key enzymes in triacylglycerol remobilization. We employed a serine hydrolase specific activity-based labeling approach in combination with quantitative proteomics to identify and rank hydrolases based on their relative activity in 11 sections of the small intestine. Moreover, we identified several clusters of enzymes showing similar activity distribution along the small intestine. Merging our activity-based results with substrate specificity and subcellular localization known from previous studies, carboxylesterase 2e and arylacetamide deacetylase emerge as promising candidates for triacylglycerol mobilization from cytosolic lipid droplets in enterocytes.
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http://dx.doi.org/10.1074/mcp.RA120.002171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710144PMC
December 2020

Intestine-specific DGAT1 deficiency improves atherosclerosis in apolipoprotein E knockout mice by reducing systemic cholesterol burden.

Atherosclerosis 2020 10 10;310:26-36. Epub 2020 Aug 10.

Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Background And Aims: Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the rate-limiting enzyme catalyzing the final step of triglyceride synthesis by esterifying a diglyceride with a fatty acid. We have previously shown that apolipoprotein E-knockout (ApoE) mice lacking Dgat1 have reduced intestinal cholesterol absorption and potentiated macrophage cholesterol efflux, and consequently, exhibit attenuated atherogenesis. However, hematopoietic Dgat1 deficiency lacked beneficial effects on atherosclerosis. Due to our recent results on the critical role of intestinal Dgat1 in murine cholesterol homeostasis, we delineated whether intestinal Dgat1 deficiency regulates atherogenesis in mice.

Methods: We generated intestine-specific Dgat1 mice on the ApoE background (iDgat1ApoE) and determined cholesterol homeostasis and atherosclerosis development.

Results: When fed a Western-type diet, iDgat1ApoE mice exhibited a substantial decrease in fasting plasma cholesterol content in ApoB-containing lipoproteins. Although lipid absorption was delayed, iDgat1ApoE mice had reduced acute and fractional cholesterol absorption coupled with an elevated fecal caloric loss. In line, increased appearance of i.v. administered [³H]cholesterol in duodena and stool of iDgat1ApoE animals suggested potentiated cholesterol elimination. Atherosclerotic lesions were markedly smaller with beneficial alterations in plaque composition as evidenced by reduced macrophage infiltration and necrotic core size despite unaltered collagen content, indicating improved plaque stability.

Conclusions: Disruption of Dgat1 activity solely in the small intestine of ApoE mice strongly decreased plasma cholesterol levels by abrogating the assimilation of dietary cholesterol, partly by reduced absorption and increased excretion. Consequently, the reduced cholesterol burden significantly attenuated atherogenesis and improved the lesion phenotype in iDgat1ApoE mice.
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http://dx.doi.org/10.1016/j.atherosclerosis.2020.07.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116265PMC
October 2020

Myeloperoxidase and Septic Conditions Disrupt Sphingolipid Homeostasis in Murine Brain Capillaries In Vivo and Immortalized Human Brain Endothelial Cells In Vitro.

Int J Mol Sci 2020 Feb 9;21(3). Epub 2020 Feb 9.

Division of Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz 8010, Austria.

During inflammation, activated leukocytes release cytotoxic mediators that compromise blood-brain barrier (BBB) function. Under inflammatory conditions, myeloperoxidase (MPO) is critically involved in inflicting BBB damage. We used genetic and pharmacological approaches to investigate whether MPO induces aberrant lipid homeostasis at the BBB in a murine endotoxemia model. To corroborate findings in a human system we studied the impact of sera from sepsis and non-sepsis patients on brain endothelial cells (hCMEC/D3). In response to endotoxin, the fatty acid, ceramide, and sphingomyelin content of isolated mouse brain capillaries dropped and barrier dysfunction occurred. In mice, genetic deficiency or pharmacological inhibition of MPO abolished these alterations. Studies in metabolic cages revealed increased physical activity and less pronounced sickness behavior of MPO compared to wild-type mice in response to sepsis. In hCMEC/D3 cells, exogenous tumor necrosis factor α (TNFα) potently regulated gene expression of pro-inflammatory cytokines and a set of genes involved in sphingolipid (SL) homeostasis. Notably, treatment of hCMEC/D3 cells with sera from septic patients reduced cellular ceramide concentrations and induced barrier and mitochondrial dysfunction. In summary, our in vivo and in vitro data revealed that inflammatory mediators including MPO, TNFα induce dysfunctional SL homeostasis in brain endothelial cells. Genetic and pharmacological inhibition of MPO attenuated endotoxin-induced alterations in SL homeostasis in vivo, highlighting the potential role of MPO as drug target to treat inflammation-induced brain dysfunction.
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http://dx.doi.org/10.3390/ijms21031143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037060PMC
February 2020

Endothelial lipase increases eNOS activating capacity of high-density lipoprotein.

Biochim Biophys Acta Mol Cell Biol Lipids 2020 04 7;1865(4):158612. Epub 2020 Jan 7.

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria; BioTechMed-Graz, Mozartgasse 12/II, 8010 Graz, Austria. Electronic address:

Endothelial lipase (EL) changes structural and functional properties of high-density lipoprotein (HDL). HDL is a relevant modulator of endothelial nitric oxide synthase (eNOS) activity, but the effect of EL on HDL induced eNOS-activation has not yet been investigated. Here, we examined the impact of EL-modified HDL (EL-HDL) on eNOS activity, subcellular trafficking, and eNOS- dependent vasorelaxation. EL-HDL and empty virus (EV)-HDL as control were isolated from human serum incubated with EL-overexpressing or EV infected HepG2 cells. EL-HDL exhibited higher capacity to induce eNOS phosphorylation at Ser1177 and eNOS activity in EA.hy 926 cells, as well as eNOS-dependent vasorelaxation of mouse aortic rings compared to control HDL. As revealed by confocal and structured illumination-microscopy EL-HDL-driven induction of eNOS was accompanied by an increased eNOS-GFP targeting to the plasma membrane and a lower eNOS-GFP colocalization with Golgi and mitochondria. Widefield microscopy of filipin stained cells revealed that EL-HDL lowered cellular free cholesterol (FC) and as found by thin-layer chromatography increased cellular cholesterol ester (CE) content. Additionally, cholesterol efflux capacity, acyl-coenzyme A: cholesterol acyltransferase activity, and HDL particle uptake were comparable between EL-HDL and control HDL. In conclusion, EL increases eNOS activating capacity of HDL, a phenomenon accompanied by an enrichment of the plasma membrane eNOS pool, a decreased cell membrane FC and increased cellular CE content.
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http://dx.doi.org/10.1016/j.bbalip.2020.158612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116681PMC
April 2020

-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

FASEB J 2019 12 1;33(12):13808-13824. Epub 2019 Nov 1.

Institute of Biochemistry, Graz University of Technology, Graz, Austria.

-acetylaspartate (NAA) is synthesized by aspartate -acetyltransferase (gene: ) from acetyl-coenzyme A and aspartate. In the brain, NAA is considered an important energy metabolite for lipid synthesis. However, the role of NAA in peripheral tissues remained elusive. Therefore, we characterized the metabolic phenotype of knockout (ko) and adipose tissue-specific (ako) -ko mice as well as NAA-supplemented mice on various diets. We identified an important role of NAA availability in the brain during adolescence, as 75% of -ko mice died on fat-free diet (FFD) after weaning but could be rescued by NAA supplementation. In adult life, NAA deficiency promotes a beneficial metabolic phenotype, as -ko and -ako mice showed reduced body weight, increased energy expenditure, and improved glucose tolerance on chow, high-fat, and FFDs. Furthermore, -deficient adipocytes exhibited increased mitochondrial respiration, ATP synthesis, and an induction of browning. Conversely, NAA-treated wild-type mice showed reduced adipocyte respiration and lipolysis and increased lipogenesis, culminating in reduced energy expenditure, glucose tolerance, and insulin sensitivity. Mechanistically, our data point to a possible role of NAA as modulator of pancreatic insulin secretion and suggest NAA as a critical energy metabolite for adipocyte and whole-body energy homeostasis.-Hofer, D. C., Zirkovits, G., Pelzmann, H. J., Huber, K., Pessentheiner, A. R., Xia, W., Uno, K., Miyazaki, T., Kon, K., Tsuneki, H., Pendl, T., Al Zoughbi, W., Madreiter-Sokolowski, C. T., Trausinger, G., Abdellatif, M., Schoiswohl, G., Schreiber, R., Eisenberg, T., Magnes, C., Sedej, S., Eckhardt, M., Sasahara, M., Sasaoka, T., Nitta, A., Hoefler, G., Graier, W. F., Kratky, D., Auwerx, J., Bogner-Strauss, J. G. -acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.
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http://dx.doi.org/10.1096/fj.201801323RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894082PMC
December 2019

Presenilin-1 Established ER-Ca Leak: a Follow Up on Its Importance for the Initial Insulin Secretion in Pancreatic Islets and β-Cells upon Elevated Glucose.

Cell Physiol Biochem 2019 ;53(3):573-586

Molecular Biology and Biochemistry, Gottfried Schatz Research Center for Cellular Signaling, Metabolism & Aging, Medical University of Graz, Graz, Austria.

Background/aims: In our recent work, the importance of GSK3β-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in β-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3β-phosphorylated presenilin-1 for responsiveness of pancreatic islets and β-cells to elevated glucose in terms of cytosolic Ca spiking and insulin secretion.

Methods: Freshly isolated pancreatic islets and the two pancreatic β-cell lines INS-1 and MIN-6 were used. Cytosolic Ca was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA.

Results: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca leak in β-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca leak. Furthermore, our data revealed that cytosolic Ca spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3β in the pancreatic β-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3β in the pancreatic β-cells and isolated pancreatic islets, respectively.

Conclusion: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and β-cells to glucose illustrated as glucose-triggered cytosolic Ca spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca leak that is due to the phosphorylation of presenilin-1 by GSK3β, a phenomenon that might be involved in the development of type 2 diabetes.
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http://dx.doi.org/10.33594/000000158DOI Listing
December 2019

ATGL/CGI-58-Dependent Hydrolysis of a Lipid Storage Pool in Murine Enterocytes.

Cell Rep 2019 08;28(7):1923-1934.e4

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, 8010 Graz, Styria, Austria; BioTechMed-Graz, 8010 Graz, Styria, Austria. Electronic address:

As circulating lipid levels are balanced by the rate of lipoprotein release and clearance from the plasma, lipid absorption in the small intestine critically contributes to the maintenance of whole-body lipid homeostasis. Within enterocytes, excessive triglycerides are transiently stored as cytosolic lipid droplets (cLDs), and their mobilization sustains lipid supply during interprandial periods. Using mice lacking adipose triglyceride lipase (ATGL) and its coactivator comparative gene identification-58 (CGI-58) exclusively in the intestine (intestine-specific double KO [iDKO]), we show that ATGL/CGI-58 are not involved in providing substrates for chylomicron synthesis. Massive intestinal cLD accumulation in iDKO mice independent of dietary lipids together with inefficient lipid incorporation into cLDs in the early absorption phase demonstrate the existence of a secretion/re-uptake cycle, corroborating the availability of two diverse cLD pools. This study identified ATGL/CGI-58 as critical players in the catabolism of basolaterally (blood) derived lipids and highlights the necessity to modify the current model of intestinal lipid metabolism.
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http://dx.doi.org/10.1016/j.celrep.2019.07.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713565PMC
August 2019

Amyloid-beta impairs insulin signaling by accelerating autophagy-lysosomal degradation of LRP-1 and IR-β in blood-brain barrier endothelial cells in vitro and in 3XTg-AD mice.

Mol Cell Neurosci 2019 09 2;99:103390. Epub 2019 Jul 2.

Division of Immunology and Pathophysiology, Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Medical University of Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Aberrant insulin signaling constitutes an early change in Alzheimer's disease (AD). Insulin receptors (IR) and low-density lipoprotein receptor-related protein-1 (LRP-1) are expressed in brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). There, insulin may regulate the function of LRP-1 in Aβ clearance from the brain. Changes in IR-β and LRP-1 and insulin signaling at the BBB in AD are not well understood. Herein, we identified a reduction in cerebral and cerebrovascular IR-β levels in 9-month-old male and female 3XTg-AD (PS1, APP, and tau) as compared to NTg mice, which is important in insulin mediated signaling responses. Reduced cerebral IR-β levels corresponded to impaired insulin signaling and LRP-1 levels in brain. Reduced cerebral and cerebrovascular IR-β and LRP-1 levels in 3XTg-AD mice correlated with elevated levels of autophagy marker LC3B. In both genotypes, high-fat diet (HFD) feeding decreased cerebral and hepatic LRP-1 expression and elevated cerebral Aβ burden without affecting cerebrovascular LRP-1 and IR-β levels. In vitro studies using primary porcine (p)BCEC revealed that Aβ peptides 1-40 or 1-42 (240 nM) reduced cellular levels and interaction of LRP-1 and IR-β thereby perturbing insulin-mediated signaling. Further mechanistic investigation revealed that Aβ treatment accelerated the autophagy-lysosomal degradation of IR-β and LRP-1 in pBCEC. LRP-1 silencing in pBCEC decreased IR-β levels through post-translational pathways further deteriorating insulin-mediated responses at the BBB. Our findings indicate that LRP-1 proves important for insulin signaling at the BBB. Cerebral Aβ burden in AD may accelerate LRP-1 and IR-β degradation in BCEC thereby contributing to impaired cerebral and cerebromicrovascular insulin effects.
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http://dx.doi.org/10.1016/j.mcn.2019.103390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6897558PMC
September 2019

Endothelial lipase increases antioxidative capacity of high-density lipoprotein.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 10 17;1864(10):1363-1374. Epub 2019 Jun 17.

Otto Loewi Research Center, Division of Physiological Chemistry, Medical University of Graz, Neue Stiftingtalstraße 6/3, 8010 Graz, Austria.

Endothelial lipase (EL) is a strong determinant of structural and functional properties of high-density lipoprotein (HDL). We examined whether the antioxidative capacity of HDL is affected by EL. EL-modified HDL (EL-HDL) and control EV-HDL were generated by incubation of HDL with EL- overexpressing or control HepG2 cells. As determined by native gradient gel electrophoresis, electron microscopy, and small-angle X-ray scattering EL-HDL is smaller than EV-HDL. Mass spectrometry revealed an enrichment of EL-HDL with lipolytic products and depletion of phospholipids and triacylglycerol. Kinetics of conjugated diene formation and HPLC-based malondialdehyde quantification revealed that EL-HDL exhibited a significantly higher resistance to copper ion-induced oxidation and a significantly higher capacity to protect low-density lipoprotein (LDL) from copper ion-induced oxidation when compared to EV-HDL. Depletion of the lipolytic products from EL-HDL abolished the capacity of EL-HDL to protect LDL from copper ion-induced oxidation, which could be partially restored by lysophosphatidylcholine enrichment. Proteomics of HDL incubated with oxidized LDL revealed significantly higher levels of methionine 136 sulfoxide in EL-HDL compared to EV-HDL. Chloramine T (oxidizes methionines and modifies free thiols), diminished the difference between EL-HDL and EV-HDL regarding the capacity to protect LDL from oxidation. In absence of LDL small EV-HDL and EL-HDL exhibited higher resistance to copper ion-induced oxidation when compared to respective large particles. In conclusion, the augmented antioxidative capacity of EL-HDL is primarily determined by the enrichment of HDL with EL-generated lipolytic products and to a lesser extent by the decreased HDL particle size and the increased activity of chloramine T-sensitive mechanisms.
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http://dx.doi.org/10.1016/j.bbalip.2019.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699986PMC
October 2019

Glycogen Synthase Kinase 3 Beta Controls Presenilin-1-Mediated Endoplasmic Reticulum Ca²⁺ Leak Directed to Mitochondria in Pancreatic Islets and β-Cells.

Cell Physiol Biochem 2019 18;52(1):57-75. Epub 2019 Feb 18.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center for Cellular Signaling, Metabolism & Aging, Medical University of Graz, Graz, Austria.

Background/aims: In pancreatic β-cells, the intracellular Ca²⁺ homeostasis is an essential regulator of the cells major functions. The endoplasmic reticulum (ER) as interactive intracellular Ca²⁺ store balances cellular Ca²⁺. In this study basal ER Ca²⁺ homeostasis was evaluated in order to reveal potential β-cell-specificity of ER Ca²⁺ handling and its consequences for mitochondrial Ca²⁺, ATP and respiration.

Methods: The two pancreatic cell lines INS-1 and MIN-6, freshly isolated pancreatic islets, and the two non-pancreatic cell lines HeLA and EA.hy926 were used. Cytosolic, ER and mitochondrial Ca²⁺ and ATP measurements were performed using single cell fluorescence microscopy and respective (genetically-encoded) sensors/dyes. Mitochondrial respiration was monitored by respirometry. GSK3β activity was measured with ELISA.

Results: An atypical ER Ca²⁺ leak was observed exclusively in pancreatic islets and β-cells. This continuous ER Ca²⁺ efflux is directed to mitochondria and increases basal respiration and organellar ATP levels, is established by GSK3β-mediated phosphorylation of presenilin-1, and is prevented by either knockdown of presenilin-1 or an inhibition/knockdown of GSK3β. Expression of a presenlin-1 mutant that mimics GSK3β-mediated phosphorylation established a β-cell-like ER Ca²⁺ leak in HeLa and EA.hy926 cells. The ER Ca²⁺ loss in β-cells was compensated at steady state by Ca²⁺ entry that is linked to the activity of TRPC3.

Conclusion: Pancreatic β-cells establish a cell-specific ER Ca²⁺ leak that is under the control of GSK3β and directed to mitochondria, thus, reflecting a cell-specific intracellular Ca²⁺ handling for basal mitochondrial activity.
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http://dx.doi.org/10.33594/000000005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459368PMC
March 2019

Intestine-Specific Overexpression of Carboxylesterase 2c Protects Mice From Diet-Induced Liver Steatosis and Obesity.

Hepatol Commun 2019 Feb 17;3(2):227-245. Epub 2018 Dec 17.

Institute of Molecular Biosciences University of Graz Graz Austria.

Murine hepatic carboxylesterase 2c () and the presumed human ortholog carboxylesterase 2 () have been implicated in the development of nonalcoholic fatty liver disease (NAFLD) in mice and obese humans. These studies demonstrated that hydrolyzes triglycerides (TGs) in hepatocytes. Interestingly, / is most abundantly expressed in the intestine, indicating a role of / in intestinal TG metabolism. Here we show that is an important enzyme in intestinal lipid metabolism in mice. Intestine-specific overexpression (Ces2c) provoked increased fatty acid oxidation (FAO) in the small intestine accompanied by enhanced chylomicron clearance from the circulation. As a consequence, high-fat diet-fed Ces2c mice were resistant to excessive diet-induced weight gain and adipose tissue expansion. Notably, intestinal overexpression increased hepatic insulin sensitivity and protected mice from NAFLD development. Although lipid absorption was not affected in Ces2c mice, fecal energy content was significantly increased. Mechanistically, we demonstrate that is a potent neutral lipase, which efficiently hydrolyzes TGs and diglycerides (DGs) in the small intestine, thereby generating fatty acids (FAs) for FAO and monoglycerides (MGs) and DGs for potential re-esterification. Consequently, the increased availability of MGs and DGs for re-esterification and primordial apolipoprotein B particle lipidation may increase chylomicron size, ultimately mediating more efficient chylomicron clearance from the circulation. This study suggests a critical role for Ces2c in intestinal lipid metabolism and highlights the importance of intestinal lipolysis to protect mice from the development of hepatic insulin resistance, NAFLD, and excessive diet-induced weight gain during metabolic stress.
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http://dx.doi.org/10.1002/hep4.1292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357831PMC
February 2019

Hepatocyte-specific lysosomal acid lipase deficiency protects mice from diet-induced obesity but promotes hepatic inflammation.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 04 9;1864(4):500-511. Epub 2019 Jan 9.

Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triglycerides (TG) to generate fatty acids (FA) and cholesterol. LAL deficiency (LAL-D) in both humans and mice leads to hepatomegaly, hypercholesterolemia, and shortened life span. Despite its essential role in lysosomal neutral lipid catabolism, the cell type-specific contribution of LAL to disease progression is still elusive. To investigate the role of LAL in the liver in more detail and to exclude the contribution of LAL in macrophages, we generated hepatocyte-specific LAL-deficient mice (Liv-Lipa) and fed them either chow or high fat/high cholesterol diets (HF/HCD). Comparable to systemic LAL-D, Liv-Lipa mice were resistant to diet-induced obesity independent of food intake, movement, and energy expenditure. Reduced body weight gain was mainly due to reduced white adipose tissue depots. Furthermore, Liv-Lipa mice exhibited improved glucose clearance during glucose and insulin tolerance tests compared to control mice. Analysis of hepatic lipid content revealed a massive reduction of TG, whereas CE concentrations were markedly increased, leading to CE crystal formation in the livers of Liv-Lipa mice. Elevated plasma transaminase activities, increased pro-inflammatory cytokines and chemokines as well as hepatic macrophage infiltration indicated liver inflammation. Our data provide evidence that hepatocyte-specific LAL deficiency is sufficient to alter whole-body lipid and energy homeostasis in mice. We conclude that hepatic LAL plays a pivotal role by preventing liver damage and maintaining lipid and energy homeostasis, especially during high lipid availability.
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http://dx.doi.org/10.1016/j.bbalip.2019.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372077PMC
April 2019

N-acetylaspartate pathway is nutrient responsive and coordinates lipid and energy metabolism in brown adipocytes.

Biochim Biophys Acta Mol Cell Res 2019 03 31;1866(3):337-348. Epub 2018 Aug 31.

Institute of Biochemistry, Graz University of Technology, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

The discovery of significant amounts of metabolically active brown adipose tissue (BAT) in adult humans renders it a promising target for anti-obesity therapies by inducing weight loss through increased energy expenditure. The components of the N-acetylaspartate (NAA) pathway are highly abundant in BAT. Aspartate N-acetyltransferase (Asp-NAT, encoded by Nat8l) synthesizes NAA from acetyl-CoA and aspartate and increases energy expenditure in brown adipocytes. However, the exact mechanism how the NAA pathway contributes to accelerated mobilization and oxidation of lipids and the physiological regulation of the NAA pathway remained elusive. Here, we demonstrate that the expression of NAA pathway genes corresponds to nutrient availability and specifically responds to changes in exogenous glucose. NAA is preferentially produced from glucose-derived acetyl-CoA and aspartate and its concentration increases during adipogenesis. Overexpression of Nat8l drains glucose-derived acetyl-CoA into the NAA pool at the expense of cellular lipids and certain amino acids. Mechanistically, we elucidated that a combined activation of neutral and lysosomal (acid) lipolysis is responsible for the increased lipid degradation. Specifically, translocation of the transcription factor EB to the nucleus activates the biosynthesis of autophagosomes and lysosomes. Lipid degradation within lysosomes accompanied by adipose triglyceride lipase-mediated lipolysis delivers fatty acids for the support of elevated mitochondrial respiration. Together, our data suggest a crucial role of the NAA pathway in energy metabolism and metabolic adaptation in BAT.
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http://dx.doi.org/10.1016/j.bbamcr.2018.08.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390944PMC
March 2019

The PPARα Agonist Fenofibrate Prevents Formation of Protein Aggregates (Mallory-Denk bodies) in a Murine Model of Steatohepatitis-like Hepatotoxicity.

Sci Rep 2018 08 28;8(1):12964. Epub 2018 Aug 28.

Institute of Pathology, Medical University of Graz, Graz, Austria.

Chronic intoxication of mice with the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to morphological and metabolic changes closely resembling steatohepatitis, a severe form of metabolic liver disease in humans. Since human steatohepatitis (both the alcoholic and non-alcoholic type) is characterized by reduced expression of PPARα and disturbed lipid metabolism we investigated the role of this ligand-activated receptor in the development of DDC-induced liver injury. Acute DDC-intoxication was accompanied by early significant downregulation of Pparα mRNA expression along with PPARα-controlled stress-response and lipid metabolism genes that persisted in the chronic stage. Administration of the specific PPARα agonist fenofibrate together with DDC prevented the downregulation of PPARα-associated genes and also improved the stress response of Nrf2-dependent redox-regulating genes. Moreover, oxidative stress and inflammation were strongly reduced by DDC/fenofibrate co-treatment. In addition, fenofibrate prevented the disruption of hepatocyte intermediate filament cytoskeleton and the formation of Mallory-Denk bodies at late stages of DDC intoxication. Our findings show that, like in human steatohepatitis, PPARα is downregulated in the DDC model of steatohepatitis-like hepatocellular damage. Its downregulation and the pathomorphologic features of steatohepatitis are prevented by co-administration of fenofibrate.
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http://dx.doi.org/10.1038/s41598-018-31389-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113278PMC
August 2018

Loss of ABHD15 Impairs the Anti-lipolytic Action of Insulin by Altering PDE3B Stability and Contributes to Insulin Resistance.

Cell Rep 2018 05;23(7):1948-1961

Institute of Biochemistry, Graz University of Technology, 8010 Graz, Austria; BioTechMed-Graz, 8010 Graz, Austria. Electronic address:

Elevated circulating fatty acids (FAs) contribute to obesity-associated metabolic complications, but the mechanisms by which insulin suppresses lipolysis are poorly understood. We show that α/β-hydrolase domain-containing 15 (ABHD15) is required for the anti-lipolytic action of insulin in white adipose tissue (WAT). Neither insulin nor glucose treatments can suppress FA mobilization in global and conditional Abhd15-knockout (KO) mice. Accordingly, insulin signaling is impaired in Abhd15-KO adipocytes, as indicated by reduced AKT phosphorylation, glucose uptake, and de novo lipogenesis. In vitro data reveal that ABHD15 associates with and stabilizes phosphodiesterase 3B (PDE3B). Accordingly, PDE3B expression is decreased in the WAT of Abhd15-KO mice, mechanistically explaining increased protein kinase A (PKA) activity, hormone-sensitive lipase (HSL) phosphorylation, and undiminished FA release upon insulin signaling. Ultimately, Abhd15-KO mice develop insulin resistance. Notably, ABHD15 expression is decreased in humans with obesity and diabetes compared to humans with obesity and normal glucose tolerance, identifying ABHD15 as a potential therapeutic target to mitigate insulin resistance.
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http://dx.doi.org/10.1016/j.celrep.2018.04.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390945PMC
May 2018

Myristic acid induces proteomic and secretomic changes associated with steatosis, cytoskeleton remodeling, endoplasmic reticulum stress, protein turnover and exosome release in HepG2 cells.

J Proteomics 2018 06 12;181:118-130. Epub 2018 Apr 12.

Department of Biotechnology, Proteomics and Mass Spectrometry Laboratory, University of Verona, Strada le Grazie 15, Verona, Italy. Electronic address:

Myristic acid, the 14-carbon saturated fatty acid (C14:0), is associated to an increased cardiovascular disease risk. Since it is found in low concentration in cells, its specific properties have not been fully analyzed. The aim of this study was to explore the cell response to this fatty acid to help explaining clinical findings on the relationship between C14:0 and cardiovascular disease. The human liver HepG2 cell line was used to investigate the hepatic response to C14:0 in a combined proteomic and secretomic approach. A total of 47 intracellular and 32 secreted proteins were deregulated after treatments with different concentrations of C14:0. Data are available via ProteomeXchange (PXD007902). In addition, C14:0 treatment of primary murine hepatocytes confirmed that C14:0 induces lipid droplet accumulation and elevates perilipin-2 levels. Functional enrichment analysis revealed that C14:0 modulates lipid droplet formation and cytoskeleton organization, induce ER stress, changes in exosome and extracellular miRNA sorting in HepG2cells. Our data provide for the first time a proteomic profiling of the effects of C14:0 in human hepatoma cells and contribute to the elucidation of molecular mechanisms through which this fatty acid may cause adverse health effects.

Biological Significance: Myristic acid is correlated with an increase in plasma cholesterol and mortality due to cardiovascular diseases. This study is the first example of an integration of proteomic and secretomic analysis of HepG2 cells to investigate the specific properties and functional roles of myristic acid on hepatic cells. Our analyses will lead to a better understanding of the myristic acid induced effects and can elicit new diagnostic and treatment strategies based on altered proteins.
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http://dx.doi.org/10.1016/j.jprot.2018.04.008DOI Listing
June 2018

Lysosomal Cholesterol Hydrolysis Couples Efferocytosis to Anti-Inflammatory Oxysterol Production.

Circ Res 2018 05 9;122(10):1369-1384. Epub 2018 Mar 9.

From the Institut National de la Santé et de la Recherche Médicale (Inserm) U1065, Université Côte d'Azur, Centre Méditerranéen de Médecine Moléculaire (C3M), Atip-Avenir, Fédération Hospitalo-Universitaire (FHU) Oncoage, Nice, France (M.V., S.I., L.-E.A., E.G., S.M., L.B., R.G., L.Y.-C.)

Rationale: Macrophages face a substantial amount of cholesterol after the ingestion of apoptotic cells, and the LIPA (lysosomal acid lipase) has a major role in hydrolyzing cholesteryl esters in the endocytic compartment.

Objective: Here, we directly investigated the role of LIPA-mediated clearance of apoptotic cells both in vitro and in vivo.

Methods And Results: We show that LIPA inhibition causes a defective efferocytic response because of impaired generation of 25-hydroxycholesterol and 27-hydroxycholesterol. Reduced synthesis of 25-hydroxycholesterol after LIPA inhibition contributed to defective mitochondria-associated membrane leading to mitochondrial oxidative stress-induced NLRP3 (NOD-like receptor family, pyrin domain containing) inflammasome activation and caspase-1-dependent Rac1 (Ras-related C3 botulinum toxin substrate 1) degradation. A secondary event consisting of failure to appropriately activate liver X receptor-mediated pathways led to mitigation of cholesterol efflux and apoptotic cell clearance. In mice, LIPA inhibition caused defective clearance of apoptotic lymphocytes and stressed erythrocytes by hepatic and splenic macrophages, culminating in splenomegaly and splenic iron accumulation under hypercholesterolemia.

Conclusions: Our findings position lysosomal cholesterol hydrolysis as a critical process that prevents metabolic inflammation by enabling efficient macrophage apoptotic cell clearance.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.312333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034181PMC
May 2018

Lysosomal acid lipase regulates fatty acid channeling in brown adipose tissue to maintain thermogenesis.

Biochim Biophys Acta Mol Cell Biol Lipids 2018 Apr 31;1863(4):467-478. Epub 2018 Jan 31.

Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and triacylglycerols in lysosomes of multiple cells and tissues. Here, we explored the role of LAL in brown adipose tissue (BAT). LAL-deficient (Lal-/-) mice exhibit markedly reduced UCP1 expression in BAT, modified BAT morphology with accumulation of lysosomes, and mitochondrial dysfunction, consequently leading to regular hypothermic events in mice kept at room temperature. Cold exposure resulted in reduced lipid uptake into BAT, thereby aggravating dyslipidemia and causing life threatening hypothermia in Lal-/- mice. Linking LAL as a potential regulator of lipoprotein lipase activity, we found Angptl4 mRNA expression upregulated in BAT. Our data demonstrate that LAL is critical for shuttling fatty acids derived from circulating lipoproteins to BAT during cold exposure. We conclude that inhibited lysosomal lipid hydrolysis in BAT leads to impaired thermogenesis in Lal-/- mice.
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http://dx.doi.org/10.1016/j.bbalip.2018.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839464PMC
April 2018

Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo.

Nat Commun 2017 11 10;8(1):1422. Epub 2017 Nov 10.

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010, Graz, Austria.

Changes in intra- and extracellular potassium ion (K) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K changes is severely limited by the lack of practicable detection methods. We developed K-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K signals. Hitherto unknown Ca-triggered, organelle-specific K changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K assays and open new avenues for live-cell K imaging.
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http://dx.doi.org/10.1038/s41467-017-01615-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681659PMC
November 2017

Impact of Endothelial Lipase on Cholesterol Efflux Capacity of Serum and High-density Lipoprotein.

Sci Rep 2017 10 2;7(1):12485. Epub 2017 Oct 2.

Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University Graz, Neue Stiftingtalstraße 6/6, 8010, Graz, Austria.

Endothelial lipase (EL) is a potent modulator of the structural and functional properties of HDL. Impact of EL on cholesterol efflux capacity (CEC) of serum and isolated HDL is not well understood and apparently contradictory data were published. Here, we systematically examined the impact of EL on composition and CEC of serum and isolated HDL, in vitro and in vivo, using EL-overexpressing cells and EL-overexpressing mice. CEC was examined in a validated assay using H-cholesterol labelled J774 macrophages. In vitro EL-modification of serum resulted in complex alterations, including enrichment of serum with lipid-free/-poor apoA-I, decreased size of human (but not mouse) HDL and altered HDL lipid composition. EL-modification of serum increased CEC, in line with increased lipid-free/-poor apoA-I formation. In contrast, CEC of isolated HDL was decreased likely through altered lipid composition. In contrast to in vitro results, EL-overexpression in mice markedly decreased HDL-cholesterol and apolipoprotein A-I serum levels associated with a decreased CEC of serum. HDL lipid composition was altered, but HDL particle size and CEC were not affected. Our study highlights the multiple and complex effects of EL on HDL composition and function and may help to clarify the seemingly contradictory data found in published articles.
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http://dx.doi.org/10.1038/s41598-017-12882-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624901PMC
October 2017

Cytosolic lipolysis and lipophagy: two sides of the same coin.

Nat Rev Mol Cell Biol 2017 Nov 30;18(11):671-684. Epub 2017 Aug 30.

BioTechMed-Graz, Mozartgasse 12, 8010 Graz, Austria.

Fatty acids are the most efficient substrates for energy production in vertebrates and are essential components of the lipids that form biological membranes. Synthesis of triacylglycerols from non-esterified free fatty acids (FFAs) combined with triacylglycerol storage represents a highly efficient strategy to stockpile FFAs in cells and prevent FFA-induced lipotoxicity. Although essentially all vertebrate cells have some capacity to store and utilize triacylglycerols, white adipose tissue is by far the largest triacylglycerol depot and is uniquely able to supply FFAs to other tissues. The release of FFAs from triacylglycerols requires their enzymatic hydrolysis by a process called lipolysis. Recent discoveries thoroughly altered and extended our understanding of lipolysis. This Review discusses how cytosolic 'neutral' lipolysis and lipophagy, which utilizes 'acid' lipolysis in lysosomes, degrade cellular triacylglycerols as well as how these pathways communicate, how they affect lipid metabolism and energy homeostasis and how their dysfunction affects the pathogenesis of metabolic diseases. Answers to these questions will likely uncover novel strategies for the treatment of prevalent metabolic diseases.
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http://dx.doi.org/10.1038/nrm.2017.76DOI Listing
November 2017

Lysosomal lipid hydrolysis provides substrates for lipid mediator synthesis in murine macrophages.

Oncotarget 2017 Jun;8(25):40037-40051

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria.

Degradation of lysosomal lipids requires lysosomal acid lipase (LAL), the only intracellular lipase known to be active at acidic pH. We found LAL to be expressed in murine immune cells with highest mRNA expression in macrophages and neutrophils. Furthermore, we observed that loss of LAL in mice caused lipid accumulation in white blood cells in the peripheral circulation, which increased in response to an acute inflammatory stimulus. Lal-deficient (-/-) macrophages accumulate neutral lipids, mainly cholesteryl esters, within lysosomes. The cholesteryl ester fraction is particularly enriched in the PUFAs 18:2 and 20:4, important precursor molecules for lipid mediator synthesis. To investigate whether loss of LAL activity affects the generation of lipid mediators and to eliminate potential systemic effects from other cells and tissues involved in the pronounced phenotype of Lal-/- mice, we treated macrophages from Wt mice with the LAL-specific inhibitor LAListat-2. Acute inhibition of LAL resulted in reduced release of 18:2- and 20:4-derived mediators from macrophages, indicating that lipid hydrolysis by LAL is an important source for lipid mediator synthesis in macrophages. We conclude that lysosomes should be considered as organelles that provide precursor molecules for lipid mediators such as eicosanoids.
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http://dx.doi.org/10.18632/oncotarget.16673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522325PMC
June 2017

Monoglyceride lipase deficiency affects hepatic cholesterol metabolism and lipid-dependent gut transit in ApoE-/- mice.

Oncotarget 2017 May;8(20):33122-33136

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria.

Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among various MG species MGL also degrades 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid and potent activator of cannabinoid receptors (CBR) 1 and 2. MGL-knockout (-/-) mice exhibit pronounced 2-AG accumulation, but lack central cannabimimetic effects due to CB1R desensitization. We have previously shown that MGL affects plaque stability in apolipoprotein E (ApoE)-/- mice, an established animal model for dyslipidemia and atherosclerosis. In the current study, we investigated functional consequences of MGL deficiency on lipid and energy metabolism in ApoE/MGL double knockout (DKO) mice. MGL deficiency affected hepatic cholesterol metabolism by causing increased cholesterol elimination via the biliary pathway. Moreover, DKO mice exhibit lipid-triggered delay in gastric emptying without major effects on overall triglyceride and cholesterol absorption. The observed phenotype of DKO mice is likely not a consequence of potentiated CB1R signaling but rather dependent on the activation of alternative signaling pathways. We conclude that MGL deficiency causes complex metabolic changes including cholesterol metabolism and regulation of gut transit independent of the endocannabinoid system.
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http://dx.doi.org/10.18632/oncotarget.16529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464855PMC
May 2017

Critical role of the peroxisomal protein PEX16 in white adipocyte development and lipid homeostasis.

Biochim Biophys Acta Mol Cell Biol Lipids 2017 Mar 23;1862(3):358-368. Epub 2016 Dec 23.

Institute of Biochemistry, Graz University of Technology, Austria. Electronic address:

The importance of peroxisomes for adipocyte function is poorly understood. Herein, we provide insights into the critical role of peroxin 16 (PEX16)-mediated peroxisome biogenesis in adipocyte development and lipid metabolism. Pex16 is highly expressed in adipose tissues and upregulated during adipogenesis of murine and human cells. We demonstrate that Pex16 is a target gene of the adipogenesis "master-regulator" PPARγ. Stable silencing of Pex16 in 3T3-L1 cells strongly reduced the number of peroxisomes while mitochondrial number was unaffected. Concomitantly, peroxisomal fatty acid (FA) oxidation was reduced, thereby causing accumulation of long- and very long-chain (polyunsaturated) FAs and reduction of odd-chain FAs. Further, Pex16-silencing decreased cellular oxygen consumption and increased FA release. Additionally, silencing of Pex16 impaired adipocyte differentiation, lipogenic and adipogenic marker gene expression, and cellular triglyceride stores. Addition of PPARγ agonist rosiglitazone and peroxisome-related lipid species to Pex16-silenced 3T3-L1 cells rescued adipogenesis. These data provide evidence that PEX16 is required for peroxisome biogenesis and highlights the relevance of peroxisomes for adipogenesis and adipocyte lipid metabolism.
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http://dx.doi.org/10.1016/j.bbalip.2016.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116240PMC
March 2017