Erika Bereczki

Apolipoprotein B100 acts as a molecular link between lipid‐induced endoplasmic reticulum stress and hepatic insulin resistance

Accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER) results in ER stress and lipid overload‐induced ER stress has been implicated in the development of insulin resistance. Here, evidence is provided for a molecular link between hepatic apolipoprotein B100 (apoB100), induction of ER stress, and attenuated insulin signaling. First, in vivo upregulation of hepatic apoB100 by a lipogenic diet was found to be closely associated with ER stress and attenuated insulin signaling in the liver. Direct in vivo overexpression of human apoB100 in a mouse transgenic model further supported the link between excessive apoB100 expression and hepatic ER stress. Human apoB100 transgenic mice exhibited hypertriglyceridemia and hyperglycemia. In vitro, accumulation of cellular apoB100 by free fatty acid (oleate) stimulation or constant expression of wild‐type or N‐glycosylation mutant apoB50 in hepatic cells induced ER stress. This led to perturbed activation of glycogen synthase kinase 3 and glycogen synthase by way of the activation of c‐Jun N‐terminal kinase and suppression of insulin signaling cascade, suggesting that dysregulation of apoB was sufficient to disturb ER homeostasis and induce hepatic insulin resistance. Small interfering (si)RNA‐mediated attenuation of elevated apoB level in the apoB50‐expressing cells rescued cells from lipid‐induced ER stress and reversed insulin insensitivity. Conclusion: These findings implicate apoB100 as a molecular link between lipid‐induced ER stress and hepatic insulin resistance. (HEPATOLOGY 2009.)

Noladin ether, a putative endocannabinoid, inhibits μ-opioid receptor activation via CB2 cannabinoid receptors

We examined the occurrence of possible changes in mRNA expression and the functional activity of opioid receptors after acute in vivo and in vitro treatment with the putative endogenous cannabinoid noladin ether. While noladin ether (NE) demonstrates agonist activity at CB1 cannabinoid receptors, recent data indicate that NE acts as a full agonist at CB2 cannabinoid receptors too. Considering the functional interactions between opioids and cannabinoids, it is of interest to examine whether NE affects the opioid system. To that end, we studied the influence of NE on mu-opioid receptor (MOR) mRNA expression and MOR mediated G-protein signaling. We used real-time PCR and [35S]GTPgammaS binding assays to examine the changes of MOR mRNA levels and the capability of the mu-opioid agonist peptide ([D-Ala2,(NMe)Phe4,Gly5-ol]enkephalin (DAMGO) in activating regulatory G-proteins via MORs in forebrain membrane fractions of wild-type (w.t., CB1+/+) and CB1 receptor deficient transgenic mice (knockout, CB1-/-). We found, that the expression of MOR mRNAs significantly decreased both in CB1+/+ and CB1-/- forebrain after a single injection of NE at 1 mg/kg when compared to control. Consequently, MOR-mediated signaling is attenuated after acute in vivo treatment with NE in both CB1+/+ and CB1-/- mice. Inhibition on MOR mediated activation is observed after in vitro NE administration as well. Radioligand binding competition studies showed that the noticed effect of NE on MOR signaling is not mediated through MORs. Both in vivo and in vitro attenuations of NE can be antagonized by the CB2 selective antagonist SR144528. Taken together, our data suggest that the NE caused pronounced decrease in the activity of MOR is mediated via CB2 cannabinoid receptors.

CB2 cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: Role of CB2 receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.

mTor is a signaling hub in cell survival: a mass-spectrometry-based proteomics investigation.

mTor plays a central role in controlling protein homeostasis and cell survival. Recently, we have demonstrated that perturbations of mTor signaling are implicated in Alzheimers disease (AD) and that mTor complex 1 (mTorC1) is involved in the formation of toxic phospho-tau. Therefore, we employed mass-spectrometry-based proteomics to identify specific protein expression changes in relation with cell survival in human neuroblastoma SH-SY5Y cells expressing genetically modified mTor. Cell death in SH-SY5Y cells was induced by moderate serum deprivation. Using flow cytometry we observed that up-regulated mTor complex 2 (mTorC2) increases the number of viable cells. By using a combination approach of proteomic and enrichment analysis we have identified several proteins (Thioredoxin-dependent peroxide reductase, Peroxiredoxin-5, Cofilin 1 (non-muscle), Annexin A5, Mortalin, and 14-3-3 protein zeta/delta) involved in mitochondrial integrity, apoptotosis, and pro-survival functions (caspase inhibitor activity and anti-apoptosis) that were significantly altered by mTor activity modulation. The major findings of this study are the implication of mTorC2 but not mTorC1 in cell viability modulation by activating the pro-survival machinery. Taken together, these results suggest that up-regulated mTorC2 might be playing an important role in promoting cell survival by suppressing the mitochondria-caspase-apoptotic pathway in vitro.

Biglycan protects cardiomyocytes against hypoxia/reoxygenation injury: role of nitric oxide.

Biglycan, a proteoglycan component of extracellular matrix, has been suspected to contribute to the development of atherosclerosis, but overexpression of biglycan in transgenic mice has been shown to induce cardioprotective genes including nitric oxide (NO) synthases in the heart. Therefore, here we hypothesized if exogenous administration of biglycan exerts cytoprotection. Primary cardiomyocytes from neonatal rats were subjected to 150 min hypoxia and 2 h reoxygenation. Mortality of cardiomyocytes was dose-dependently attenuated by pretreatment with 1-100 nM biglycan. Biglycan enhanced eNOS mRNA and protein, and significantly increased NO content of cardiomyocytes. The NO synthase inhibitor l-nitro-arginine-methyl-ester significantly attenuated the cytoprotective effect of biglycan. This is the first demonstration that biglycan leads to cytoprotection against hypoxia/reoxygenation injury, and that this phenomenon is partially mediated by an NO-dependent mechanism.

Human apoB overexpression and a high-cholesterol diet differently modify the brain APP metabolism in the transgenic mouse model of atherosclerosis

Epidemiological and biochemical data suggest a link between the cholesterol metabolism, the amyloid precursor protein (APP) processing and the increased cerebral beta-amyloid (Abeta) deposition in Alzheimers disease (AD). The individual and combined effects of a high-cholesterol (HC) diet and the overexpression of the human apoB-100 gene were therefore examined on the cerebral expression and processing of APP in homozygous apoB-100 transgenic mice [Tg (apoB(+/+))], a validated model of atherosclerosis. When fed with 2% cholesterol for 17 weeks, only the wild-type mice exhibited significantly increased APP695 (123%) and APP770 (138%) mRNA levels in the cortex. The HC diet-induced hypercholesterolemia significantly increased the APP isoform levels in the membrane-bound fraction, not only in the wild-type animals (114%), but also in the Tg apoB(+/+) group (171%). The overexpression of human apoB-100 gene by the liver alone reduced the brain APP isoform levels in the membrane-bound fraction (78%), whereas the levels were increased by the combined effect of HC and the overexpression of the human apoB-100 gene (134%). The protein kinase C and beta-secretase protein levels were not altered by the individual or combined effects of these two factors. Our data indicate that the two atherogenic factors, the HC diet and the overexpression of the human apoB-100 gene by the liver, could exert different effects on the processing and expression of APP in the mice brain.

Overexpression of human apolipoprotein B-100 induces severe neurodegeneration in transgenic mice.

Recent studies showed correlation between increased serum apolipoprotein B-100 (apoB-100) level and Alzheimers disease. To reveal the possible role of apoB-100 in neurodegeneration, we analyzed the serum lipoprotein and cerebral protein profiles, amyloid plaque formation, apoptosis and brain morphology of transgenic mice overexpressing the human apoB-100 protein. Serum lipoprotein profile showed significant increase of the plasma triglyceride level, while no alteration in total cholesterol was detected. The antibody microarray experiment revealed upregulation of several cytoskeletal, neuronal proteins and proteins that belong to the mitogen activated protein kinase pathway, indicating active apoptosis in the brain. Histochemical experiments showed formation of amyloid plaques and extensive neuronal death. Biochemical changes severely affected brain morphology; a dramatic genotype-dependent enlargement of the third and lateral ventricles in the brain was detected. On the basis of earlier and present results, we conclude that overexpressed human apoB-100 protein significantly increases the level of serum lipids (triglyceride upon normal chow diet and cholesterol on cholesterol-rich diet) which leads to cerebrovascular lesions and subsequently induces apoptosis and neurodegeneration.

Bruch's membrane changes in transgenic mice overexpressing the human biglycan and apolipoprotein b-100 genes

Age-Related Macular Degeneration (AMD) is characterized by the accumulation of lipid- and protein-rich deposits in Bruchs Membrane (BrM). A consequent decrease in hydraulic conductivity and impairment of transport through BrM may play a central role in the pathogenesis of AMD. The mechanism of deposit formation in AMD had been suggested to show similarities to the formation of atherosclerotic plaques in which the interactions of extracellular matrix proteoglycans with apolipoprotein-B 100 (apoB-100) play an important role. A prime candidate for this interaction is the small leucin-rich proteoglycan biglycan. The aim of our study was to test the effect of the simultaneous overexpression of human apoB-100 and biglycan genes in combination with a high-cholesterol diet on BrM morphology in transgenic mice. Six-weeks-old homozygous apoB-100 or biglycan, hemizygous apoB-100/biglycan transgenic and wild-type C57Bl/6 mice were fed either a standard chow or a diet supplemented with 2% cholesterol for 17 weeks. Animals were sacrificed, serum lipid levels were measured and eyes were processed for transmission electron microscopy (TEM) according to standard protocol. Morphometric analysis of digitally acquired TEM images of BrM showed that in apoB-100 and double transgenic animals fed a high-cholesterol diet, the BrM thickness was significantly increased compared to wild-type animals. Both groups had electron-lucent profiles in clusters, scattered throughout the collagenous layers of BrM, and focal nodules of an amorphous material of intermediate electron-density between the plasma and basement membranes of the retinal pigment epithelium (RPE). BrM thickness in these two groups correlated well with elevated cholesterol levels. Unexpectedly, animals overexpressing biglycan alone showed a marked, diet-independent increase in BrM thickness associated with a layer of a basement membrane-like material in outer BrM. The effects of biglycan overexpression are intriguing and further investigations are needed to elucidate the underlying mechanisms.

The Role of Biglycan in the Heart

Biglycan, a member of the small leucine rich proteoglycan family, is known to be expressed in almost every tissue of our body. Although there are increasing amount of data on the biological role of biglycan, its cardiac function is still not totally clarified. Cardiac protein profiling of biglycan transgenic mice and other studies revealed its involvement in heart failure, myocardial remodeling, and a possible role in promoting cardioprotection. The localization of biglycan on the cell surface and its “pericellular” arrangement as well as the presence of reactive GAG chains on its surface suggest an involvement in transmission of extracellular signals to intracellular signaling molecules and a role in regulation of Ca++ trafficking. In this review, the role of biglycan in the heart under normal physiological as well as pathological conditions is summarized and critically discussed.

Capillary injury in the ischemic brain of hyperlipidemic, apolipoprotein B-100 transgenic mice.

AIMS Apolipoprotein B-100 (apoB-100) has been implicated in hyperlipidemia, which contributes to the pathogenesis of vascular disorders. Our aim was to investigate whether the expression of human apoB-100 in transgenic mice and/or a high-cholesterol diet cause cerebral microvascular lesions, and whether these conditions augment ischemia-related capillary damage. MAIN METHODS Human apoB-100 overexpressing transgenic (Tg(apoB-100), n=23) and wild-type mice (C5/B6, Wt, n=26) were supplied with standard or 2% cholesterol-enriched diet for 17-19 weeks. Cerebral ischemia was induced by unilateral common carotid artery occlusion. Cortical samples were embedded for electron microscopy. Microvascular density (number of microvascular profiles/examined area), lumen diameter, the swelling of astrocytic endfeet, the occurrence of endothelial microvilli (affected capillaries expressed as ratio of all capillaries encountered), and the ratio of intact capillaries (devoid of all the above pathology) were calculated. KEY FINDINGS The expression of apoB-100 coincided with decreased cortical microvascular density (195+/-7 vs. 223+/-8 vessels/mm(2), vs. Wt; P<0.008) and increased capillary lumen diameter (3.16+/-0.5 vs. 2.88+/-0.6 microm, vs. Wt; P<0.001). Cerebral ischemia promoted the swelling of perivascular astrocytes (62.1+/-4.2 vs. 36.5+/-4.0%, vs. contralateral, Wt; P<0.001), and reduced the ratio of intact capillaries (32.1+/-5.6 vs. 65.2+/-3.7%, vs. contralateral, Wt; P<0.001). Hyperlipidemia did not exacerbate the injury. SIGNIFICANCE The overexpression of human apoB-100 alters the density of the microvascular network and the diameter of capillaries, which may compromise cerebrovascular reactivity during ischemia.