Jörg Heeren

Role for LAMP‐2 in endosomal cholesterol transport

The mechanisms of endosomal and lysosomal cholesterol traffic are still poorly understood. We showed previously that unesterified cholesterol accumulates in the late endosomes and lysosomes of fibroblasts deficient in both lysosome associated membrane protein‐2 (LAMP‐2) and LAMP‐1, two abundant membrane proteins of late endosomes and lysosomes. In this study we show that in cells deficient in both LAMP‐1 and LAMP‐2 (LAMP−/−), low‐density lipoprotein (LDL) receptor levels and LDL uptake are increased as compared to wild‐type cells. However, there is a defect in esterification of both endogenous and LDL cholesterol. These results suggest that LAMP−/− cells have a defect in cholesterol transport to the site of esterification in the endoplasmic reticulum, likely due to defective export of cholesterol out of late endosomes or lysosomes. We also show that cholesterol accumulates in LAMP‐2 deficient liver and that overexpression of LAMP‐2 retards the lysosomal cholesterol accumulation induced by U18666A. These results point to a critical role for LAMP‐2 in endosomal/lysosomal cholesterol export. Moreover, the late endosomal/lysosomal cholesterol accumulation in LAMP−/− cells was diminished by overexpression of any of the three isoforms of LAMP‐2, but not by LAMP‐1. The LAMP‐2 luminal domain, the membrane‐proximal half in particular, was necessary and sufficient for the rescue effect. Taken together, our results suggest that LAMP‐2, its luminal domain in particular, plays a critical role in endosomal cholesterol transport and that this is distinct from the chaperone‐mediated autophagy function of LAMP‐2.

Caloric restriction and intermittent fasting alter hepatic lipid droplet proteome and diacylglycerol species and prevent diabetes in NZO mice

Caloric restriction and intermittent fasting are known to improve glucose homeostasis and insulin resistance in several species including humans. The aim of this study was to unravel potential mechanisms by which these interventions improve insulin sensitivity and protect from type 2 diabetes. Diabetes-susceptible New Zealand Obese mice were either 10% calorie restricted (CR) or fasted every other day (IF), and compared to ad libitum (AL) fed control mice. AL mice showed a diabetes prevalence of 43%, whereas mice under CR and IF were completely protected against hyperglycemia. Proteomic analysis of hepatic lipid droplets revealed significantly higher levels of PSMD9 (co-activator Bridge-1), MIF (macrophage migration inhibitor factor), TCEB2 (transcription elongation factor B (SIII), polypeptide 2), ACY1 (aminoacylase 1) and FABP5 (fatty acid binding protein 5), and a marked reduction of GSTA3 (glutathione S-transferase alpha 3) in samples of CR and IF mice. In addition, accumulation of diacylglycerols (DAGs) was significantly reduced in livers of IF mice (P=0.045) while CR mice showed a similar tendency (P=0.062). In particular, 9 DAG species were significantly reduced in response to IF, of which DAG-40:4 and DAG-40:7 also showed significant effects after CR. This was associated with a decreased PKCε activation and might explain the improved insulin sensitivity. In conclusion, our data indicate that protection against diabetes upon caloric restriction and intermittent fasting associates with a modulation of lipid droplet protein composition and reduction of intracellular DAG species.

Dicer1-miR-328-Bace1 signalling controls brown adipose tissue differentiation and function

Activation of brown adipose tissue (BAT) controls energy homeostasis in rodents and humans and has emerged as an innovative strategy for the treatment of obesity and type 2 diabetes mellitus. Here we show that ageing- and obesity-associated dysfunction of brown fat coincides with global microRNA downregulation due to reduced expression of the microRNA-processing node Dicer1. Consequently, heterozygosity of Dicer1 in BAT aggravated diet-induced-obesity (DIO)-evoked deterioration of glucose metabolism. Analyses of differential microRNA expression during preadipocyte commitment and mouse models of progeria, longevity and DIO identified miR-328 as a regulator of BAT differentiation. Reducing miR-328 blocked preadipocyte commitment, whereas miR-328 overexpression instigated BAT differentiation and impaired muscle progenitor commitment—partly through silencing of the β-secretase Bace1. Loss of Bace1 enhanced brown preadipocyte specification in vitro and was overexpressed in BAT of obese and progeroid mice. In vivo Bace1 inhibition delayed DIO-induced weight gain and improved glucose tolerance and insulin sensitivity. These experiments reveal Dicer1–miR-328–Bace1 signalling as a determinant of BAT function, and highlight the potential of Bace1 inhibition as a therapeutic approach to improve not only neurodegenerative diseases but also ageing- and obesity-associated impairments of BAT function.

Intraperitoneal Injection Improves the Uptake of Nanoparticle-Labeled High-Density Lipoprotein to Atherosclerotic Plaques Compared With Intravenous Injection A Multimodal Imaging Study in ApoE Knockout Mice

Background—The aim of this study was to assess whether high-density lipoprotein (HDL) labeled with superparamagnetic iron oxide nanoparticles (SPIOs) and quantum dots was able to detect atherosclerotic lesions in mice after intravenous and intraperitoneal injection by multimodal imaging. Methods and Results—Nanoparticle-labeled HDLs (NP-HDLs) were characterized in vitro by dynamic light scattering and size exclusion chromatography with subsequent cholesterol and fluorescence measurements. For biodistribution and blood clearance studies, NP-HDLSPIOs radiolabeled with 59Fe (NP-HDL59Fe-SPIOs) were injected intravenously or intraperitoneally into ApoE knockout mice (n=6), and radioactivity was measured using a gamma counter. NP-HDL accumulation within atherosclerotic plaques in vivo and ex vivo was estimated by MRI at 7 Tesla, ex vivo confocal fluorescence microscopy, x-ray fluorescence microscopy, and histological analysis (n=3). Statistical analyses were performed using a 2-tailed Student t-test. In vitro characterization of NP-HDL confirmed properties similar to endogenous HDL. Blood concentration time curves showed a biexponential decrease for the intravenous injection, whereas a slow increase followed by a steady state was noted for intraperitoneal injection. Radioactivity measurements showed predominant accumulation in the liver and spleen after both application approaches. NP-HDL59Fe-SPIOs uptake into atherosclerotic plaques increased significantly after intraperitoneal compared with intravenous injection (P<0.01). In vivo MRI showed an increased uptake of NP-HDL into atherosclerotic lesions after intraperitoneal injection, which was confirmed by ex vivo MRI, x-ray fluorescence microscopy, confocal fluorescence microscopy, and histological analysis. Conclusions—In vivo MRI and ex vivo multimodal imaging of atherosclerotic plaque using NP-HDL is feasible, and intraperitoneal application improves the uptake within vessel wall lesions compared with intravenous injection.

Endocannabinoid regulation in white and brown adipose tissue following thermogenic activation

The endocannabinoids and their main receptor, cannabinoid type-1 (CB1), suppress intracellular cyclic AMP levels and have emerged as key players in the control of energy metabolism. CB1 agonists and blockers have been reported to influence the thermogenic function of white and brown adipose tissue (WAT and BAT), affecting body weight through the inhibition and stimulation of energy expenditure, respectively. The purpose of the current study was to investigate the regulation of the endocannabinoid system in WAT and BAT following exposure to either cold or specific agonism of β3-adrenoceptors using CL316,243 (CL), conditions known to cause BAT activation and WAT browning. To address this question, we performed quantitative PCR-based mRNA profiling of genes important for endocannabinoid synthesis, degradation, and signaling, and determined endocannabinoid levels by LC-MS in WAT and BAT of control, cold-exposed, and CL-treated wild-type mice as well as primary brown adipocytes. Treatment with CL and exposure to cold caused an upregulation of endocannabinoid levels and biosynthetic enzymes in WAT. Acute β3-adrenoceptor activation increased endocannabinoids and a subset of genes of biosynthesis in BAT and primary brown adipocytes. We suggest that the cold-mediated increase in endocannabinoid tone is part of autocrine negative feed-back mechanisms controlling β3-adrenoceptor-induced BAT activation and WAT browning.

The distribution and degradation of radiolabeled superparamagnetic iron oxide nanoparticles and quantum dots in mice

Summary 51Cr-labeled, superparamagnetic, iron oxide nanoparticles (51Cr-SPIOs) and 65Zn-labeled CdSe/CdS/ZnS-quantum dots (65Zn-Qdots) were prepared using an easy, on demand, exchange-labeling technique and their particokinetic parameters were studied in mice after intravenous injection. The results indicate that the application of these heterologous isotopes can be used to successfully mark the nanoparticles during initial distribution and organ uptake, although the 65Zn-label appeared not to be fully stable. As the degradation of the nanoparticles takes place, the individual transport mechanisms for the different isotopes must be carefully taken into account. Although this variation in transport paths can bring new insights with regard to the respective trace element homeostasis, it can also limit the relevance of such trace material-based approaches in nanobioscience. By monitoring 51Cr-SPIOs after oral gavage, the gastrointestinal non-absorption of intact SPIOs in a hydrophilic or lipophilic surrounding was measured in mice with such high sensitivity for the first time. After intravenous injection, polymer-coated, 65Zn-Qdots were mainly taken up by the liver and spleen, which was different from that of ionic 65ZnCl2. Following the label for 4 weeks, an indication of substantial degradation of the nanoparticles and the release of the label into the Zn pool was observed. Confocal microscopy of rat liver cryosections (prepared 2 h after intravenous injection of polymer-coated Qdots) revealed a colocalization with markers for Kupffer cells and liver sinusoidal endothelial cells (LSEC), but not with hepatocytes. In J774 macrophages, fluorescent Qdots were found colocalized with lysosomal markers. After 24 h, no signs of degradation could be detected. However, after 12 weeks, no fluorescent nanoparticles could be detected in the liver cryosections, which would confirm our 65Zn data showing a substantial degradation of the polymer-coated CdSe/CdS/ZnS-Qdots in the liver.

Duck Hepatitis B Virus Requires Cholesterol for Endosomal Escape during Virus Entry

ABSTRACT The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.

Intraperitoneal Injection Improves the Uptake of Nanoparticle Labeled HDL to Atherosclerotic Plaques Compared to Intravenous Injection: A Multimodal Imaging Study in ApoE-/- Mice

Background—The aim of this study was to assess whether high-density lipoprotein (HDL) labeled with superparamagnetic iron oxide nanoparticles (SPIOs) and quantum dots was able to detect atherosclerotic lesions in mice after intravenous and intraperitoneal injection by multimodal imaging. Methods and Results—Nanoparticle-labeled HDLs (NP-HDLs) were characterized in vitro by dynamic light scattering and size exclusion chromatography with subsequent cholesterol and fluorescence measurements. For biodistribution and blood clearance studies, NP-HDLSPIOs radiolabeled with 59Fe (NP-HDL59Fe-SPIOs) were injected intravenously or intraperitoneally into ApoE knockout mice (n=6), and radioactivity was measured using a gamma counter. NP-HDL accumulation within atherosclerotic plaques in vivo and ex vivo was estimated by MRI at 7 Tesla, ex vivo confocal fluorescence microscopy, x-ray fluorescence microscopy, and histological analysis (n=3). Statistical analyses were performed using a 2-tailed Student t-test. In vitro characterization of NP-HDL confirmed properties similar to endogenous HDL. Blood concentration time curves showed a biexponential decrease for the intravenous injection, whereas a slow increase followed by a steady state was noted for intraperitoneal injection. Radioactivity measurements showed predominant accumulation in the liver and spleen after both application approaches. NP-HDL59Fe-SPIOs uptake into atherosclerotic plaques increased significantly after intraperitoneal compared with intravenous injection (P<0.01). In vivo MRI showed an increased uptake of NP-HDL into atherosclerotic lesions after intraperitoneal injection, which was confirmed by ex vivo MRI, x-ray fluorescence microscopy, confocal fluorescence microscopy, and histological analysis. Conclusions—In vivo MRI and ex vivo multimodal imaging of atherosclerotic plaque using NP-HDL is feasible, and intraperitoneal application improves the uptake within vessel wall lesions compared with intravenous injection.

Pharmacological Characterization of 1-Nitrosocyclohexyl Acetate, a Long-Acting Nitroxyl Donor That Shows Vasorelaxant and Antiaggregatory Effects

Nitroxyl (HNO) donors have potential benefit in the treatment of heart failure and other cardiovascular diseases. 1-Nitrosocyclohexyl acetate (NCA), a new HNO donor, in contrast to the classic HNO donors Angeli’s salt and isopropylamine NONOate, predominantly releases HNO and has a longer half-life. This study investigated the vasodilatative properties of NCA in isolated aortic rings and human platelets and its mechanism of action. NCA was applied on aortic rings isolated from wild-type mice and apolipoprotein E–deficient mice and in endothelial-denuded aortae. The mechanism of action of HNO was examined by applying NCA in the absence and presence of the HNO scavenger glutathione (GSH) and inhibitors of soluble guanylyl cyclase (sGC), adenylyl cyclase (AC), calcitonin gene-related peptide receptor (CGRP), and K+ channels. NCA induced a concentration-dependent relaxation (EC50, 4.4 µM). This response did not differ between all groups, indicating an endothelium-independent relaxation effect. The concentration-response was markedly decreased in the presence of excess GSH; the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide had no effect. Inhibitors of sGC, CGRP, and voltage-dependent K+ channels each significantly impaired the vasodilator response to NCA. In contrast, inhibitors of AC, ATP-sensitive K+ channels, or high-conductance Ca2+-activated K+ channels did not change the effects of NCA. NCA significantly reduced contractile response and platelet aggregation mediated by the thromboxane A2 mimetic 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α in a cGMP-dependent manner. In summary, NCA shows vasoprotective effects and may have a promising profile as a therapeutic agent in vascular dysfunction, warranting further evaluation.

Quantitative Activity Measurements of Brown Adipose Tissue at 7 T Magnetic Resonance Imaging After Application of Triglyceride-Rich Lipoprotein 59Fe-Superparamagnetic Iron Oxide Nanoparticle: Intravenous Versus Intraperitoneal Approach.

ObjectivesThe aim of this study was to determine metabolic activity of brown adipose tissue (BAT) with in vivo magnetic resonance imaging (MRI) after intravenous (IV) and intraperitoneal (IP) injection of radioactively labeled superparamagnetic iron oxide nanoparticles (SPIOs) embedded into a lipoprotein layer. Materials and Methods59Fe-labeled SPIOs were either polymer-coated or embedded into the lipid core of triglyceride-rich lipoproteins (TRL-59Fe-SPIOs). First biodistribution and blood half time analysis in thermoneutral mice after IP injection of either TRL-59Fe-SPIOs or polymer-coated 59Fe-SPIOs (n = 3) were performed. In the next step, cold-exposed (24 hours), BAT-activated mice (n = 10), and control thermoneutral mice (n = 10) were starved for 4 hours before IP (n = 10) or IV (n = 10) injection of TRL-59Fe-SPIOs. In vivo MRI was performed before and 24 hours after the application of the particles at a 7 T small animal MRI scanner using a T2*-weighted multiecho gradient echo sequence. R2* and &Dgr;R2* were estimated in the liver, BAT, and muscle. The biodistribution of polymer-coated 59Fe-SPIOs and TRL-59Fe-SPIOs was analyzed ex vivo using a sensitive, large-volume Hamburg whole-body radioactive counter. The amount of 59Fe-SPIOs in the liver, BAT, and muscle was correlated with the MRI measurements using the Pearson correlation coefficient. Tissue uptake of 59Fe-SPIOs was confirmed by histological and transmission electron microscopy analyses. ResultsTriglyceride-rich lipoprotein 59Fe-SPIOs exhibited a higher blood concentration after IP injection (10.1% ± 0.91% after 24 hours) and a greater [INCREMENT]R2* in the liver (103 ± 5.0 s−1), while polymer-coated SPIOs did not increase substantially in the blood stream (0.19% ± 0.01% after 24 hours; P < 0.001) and the liver (57 ± 4.08 s−1; P < 0.001). In BAT activity studies, significantly higher uptake of TRL-59Fe-SPIOs was detected in the BAT of cold-exposed mice, with [INCREMENT]R2* of 107 ± 5.5 s−1 after IV application (control mice: [INCREMENT]R2* of 22 ± 5.8 s−1; P < 0.001) and 45 ± 5.5 s−1 after IP application (control mice: [INCREMENT]R2* of 11 ± 2.9 s−1; P < 0.01). 59Fe radioactivity measurements and [INCREMENT]R2* values correlated strongly in BAT (r > 0.85; P < 0.001) and liver tissue (r > 0.85; P < 0.001). Histological and transmission electron microscopy analyses confirmed the uptake of TRL-59Fe-SPIOs within the liver and BAT for both application approaches. ConclusionsTriglyceride-rich lipoprotein–embedded SPIOs were able to escape the abdominal cavity barrier, whereas polymer-coated SPIOs did not increase substantially in the blood stream. Brown adipose tissue activity can be determined via MRI using TRL-59Fe-SPIOs. The quantification of [INCREMENT]R2* using TRL-59Fe-SPIOs is feasible and may serve as a noninvasive tool for the quantitative estimation of BAT activity.