ApoA-I Protects Pancreatic ß-Cells From Cholesterol-Induced Mitochondrial Damage and Restores Their Ability to Secrete Insulin.

BACKGROUND: High cholesterol levels in pancreatic ß-cells cause oxidative stress and decrease insulin secretion. ß-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves ß-cell insulin secretion by reducing oxidative stress. METHODS: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-ß-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by ß-cells was monitored by flow cytometry. The effects of apoA-I internalization on ß-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the ß-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in ß-cells and isolated islets with MitoSOX and confocal microscopy. RESULTS: An F1-ATPase ß-subunit on the ß-cell surface was identified as the main apoA-I-binding partner. ß-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F1-ATPase ß-subunit-dependent. ß-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase ß-subunit levels than ß-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E ß-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function. CONCLUSIONS: These results establish that ß-cells are functionally heterogeneous, and apoA-I restores insulin secretion in ß-cells with elevated cholesterol levels by improving mitochondrial redox balance.

Identification of Native and Posttranslationally Modified HLA-B*57:01-Restricted HIV Envelope Derived Epitopes Using Immunoproteomics.

The recognition of pathogen-derived peptides by T lymphocytes is the cornerstone of adaptive immunity, whereby intracellular antigens are degraded in the cytosol and short peptides assemble with class I human leukocyte antigen (HLA) molecules in the ER. These peptide-HLA complexes egress to the cell surface and are scrutinized by cytotoxic CD8+ T-cells leading to the eradication of the infected cell. Here, naturally presented HLA-B*57:01 bound peptides derived from the envelope protein of the human immunodeficiency virus (HIVenv) are identified. HIVenv peptides are present at a very small percentage of the overall HLA-B*57:01 peptidome (<0.1%) and both native and posttranslationally modified forms of two distinct HIV peptides are identified. Notably, a peptide bearing a natively encoded C-terminal tryptophan residue is also present in a modified form containing a kynurenine residue. Kynurenine is a major product of tryptophan catabolism and is abundant during inflammation and infection. Binding of these peptides at a molecular level and their immunogenicity in preliminary functional studies are examined. Modest immune responses are observed to the modified HIVenv peptide, highlighting a potential role for kynurenine-modified peptides in the immune response to HIV and other viral infections.

Arrested Hematopoiesis and Vascular Relaxation Defects in Mice with a Mutation in Dhfr.

Dihydrofolate reductase (DHFR) is a critical enzyme in the folate metabolism pathway and also plays a role in regulating nitric oxide (NO) signaling in endothelial cells. Although both coding and noncoding mutations with phenotypic effects have been identified in the human DHFR gene, no mouse model is currently available to study the consequences of perturbing DHFR in vivo In order to identify genes involved in definitive hematopoiesis, we performed a forward genetic screen and produced a mouse line, here referred to as Orana, with a point mutation in the Dhfr locus leading to a Thr136Ala substitution in the DHFR protein. Homozygote Orana mice initiate definitive hematopoiesis, but expansion of progenitors in the fetal liver is compromised, and the animals die between embryonic day 13.5 (E13.5) and E14.5. Heterozygote Orana mice survive to adulthood but have tissue-specific alterations in folate abundance and distribution, perturbed stress erythropoiesis, and impaired endothelium-dependent relaxation of the aorta consistent with the role of DHFR in regulating NO signaling. Orana mice provide insight into the dual roles of DHFR and are a useful model for investigating the role of environmental and dietary factors in the context of vascular defects caused by altered NO signaling.

Apolipoprotein A-I increases insulin secretion and production from pancreatic ß-cells via a G-protein-cAMP-PKA-FoxO1-dependent mechanism.

OBJECTIVE: Therapeutic interventions that increase plasma levels of high-density lipoproteins and apolipoprotein A-I (apoA-I) A-I, the major high-density lipoprotein apolipoprotein, improve glycemic control in people with type 2 diabetes mellitus. High-density lipoproteins and apoA-I also enhance insulin synthesis and secretion in isolated pancreatic islets and clonal ß-cell lines. This study identifies the signaling pathways that mediate these effects. APPROACH AND RESULTS: Incubation with apoA-I increased cAMP accumulation in Ins-1E cells in a concentration-dependent manner. The increase in cAMP levels was inhibited by preincubating the cells with the cell-permeable, transmembrane adenylate cyclase inhibitor, 2'5' dideoxyadenosine, but not with KH7, which inhibits soluble adenylyl cyclases. Incubation of Ins-1E cells with apoA-I resulted in colocalization of ATP-binding cassette transporter A1 with the Gαs subunit of a heterotrimeric G-protein and a Gαs subunit-dependent increase in insulin secretion. Incubation of Ins-1E cells with apoA-I also increased protein kinase A phosphorylation and reduced the nuclear localization of forkhead box protein O1 (FoxO1). Preincubation of Ins-1E cells with the protein kinase A-specific inhibitors, H89 and PKI amide, prevented apoA-I from increasing insulin secretion and mediating the nuclear exclusion of FoxO1. Transfection of Ins-1E cells with a mutated FoxO1 that is restricted to the nucleus confirmed the requirement for FoxO1 nuclear exclusion by blocking insulin secretion in apoA-I-treated Ins-1E cells. ApoA-I also increased Irs1, Irs2, Ins1, Ins2, and Pdx1 mRNA levels. CONCLUSIONS: ApoA-I increases insulin synthesis and secretion via a heterotrimeric G-protein-cAMP-protein kinase A-FoxO1-dependent mechanism that involves transmembrane adenylyl cyclases and increased transcription of key insulin response and ß-cell survival genes.

Superoxide dismutase deficiency impairs olfactory sexual signaling and alters bioenergetic function in mice.

Oxidative stress (an overproduction of reactive oxygen species in relation to defense mechanisms) may restrict investment in life history traits, such as growth, reproduction, lifespan, and the production of sexual signals to attract mates. The constraint on sexual signaling by oxidative stress is of particular interest because it has been proposed as a mechanism ensuring that only good-quality males produce the most attractive sexual signals. Despite these predictions, evidence supporting this theory is, at best, equivocal. We used a superoxide dismutase knockout mouse to demonstrate that oxidative stress directly impairs investment in morphological (preputial glands) and molecular (major urinary proteins) components of olfactory signaling essential for mate attraction. By maintaining males in a much more competitive environment than usual for mouse laboratory experiments, we also revealed a range of phenotypes of superoxide dismutase deficiency not observed in previous studies of this mouse model. This range included impaired bioenergetic function, which was undetectable in the control environment of this study. We urge further examination of model organisms in seminatural conditions and more competitive laboratory environments, as important phenotypes can be exposed under these more demanding conditions.

Accumulation of serum lipids by vascular smooth muscle cells involves a macropinocytosis-like uptake pathway and is associated with the downregulation of the ATP-binding cassette transporter A1.

Vascular smooth muscle cells (VSMC) are present in arterial intima before atherosclerotic plaques develop and are likely to be exposed to unmodified serum lipids as they enter the vessel wall. We examined the effects of sera from mice on the morphology and function of mouse VSMC. Incubation of a mouse VSMC line (MOVAS) with sera from normocholesterolemic (C57BL/6J) or hypercholesterolemic (APOE(-/-)) mice caused concentration-dependent increases in lipid accumulation as measured by AdipoRed, with the extent of lipid uptake significantly greater with the latter sera type. Inhibition of c-Jun N-terminal kinases (SP600125), Src kinases (AG1879), and clathrin-dependent endocytosis (monodansylcadaverine) to disrupt scavenger receptor-mediated uptake of lipids had no effect on serum-induced lipid accumulation by VSMC. By contrast, inhibition of macropinocytosis with antagonists of PI-3 kinase (LY294002) and actin (cytochalasin D) markedly reduced lipid accumulation. Serum exposure reduced the expression of the ATP-binding cassette transporter A1, consistent with impaired cholesterol efflux, but had no effect on the expression of markers of VSMC differentiation. Moreover, the expression of several inflammation and foam cell markers was unchanged (CCL2, CCL5, and CD68) by mouse sera. The accumulation of unmodified serum lipids by VSMC involves a macropinocytosis-like uptake pathway and is associated with the downregulation of the ATP-binding cassette transporter. We speculate that VSMC may play an atheroprotective role in arterial intima by acting as a "sink" for unmodified lipids.

Endogenous progesterone levels and frontotemporal dementia: modulation of TDP-43 and Tau levels in vitro and treatment of the A315T TARDBP mouse model.

Frontotemporal dementia (FTD) is associated with motor neurone disease (FTD-MND), corticobasal syndrome (CBS) and progressive supranuclear palsy syndrome (PSPS). Together, this group of disorders constitutes a major cause of young-onset dementia. One of the three clinical variants of FTD is progressive nonfluent aphasia (PNFA), which is focused on in this study. The steroid hormone progesterone (PROG) is known to have an important role as a neurosteroid with potent neuroprotective and promyelination properties. In a case-control study of serum samples (39 FTD, 91 controls), low serum PROG was associated with FTD overall. In subgroup analysis, low PROG levels were significantly associated with FTD-MND and CBS, but not with PSPS or PNFA. PROG levels of >195 pg/ml were significantly correlated with lower disease severity (frontotemporal dementia rating scale) for individuals with CBS. In the human neuroblastoma SK-N-MC cell line, exogenous PROG (9300-93,000 pg/ml) had a significant effect on overall Tau and nuclear TDP-43 levels, reducing total Tau levels by ∼1.5-fold and increasing nuclear TDP-43 by 1.7- to 2.0-fold. Finally, elevation of plasma PROG to a mean concentration of 5870 pg/ml in an Ala315Thr (A315T) TARDBP transgenic mouse model significantly reduced the rate of loss of locomotor control in PROG-treated, compared with placebo, mice. The PROG treatment did not significantly increase survival of the mice, which might be due to the limitation of the transgenic mouse to accurately model TDP-43-mediated neurodegeneration. Together, our clinical, cellular and animal data provide strong evidence that PROG could be a valid therapy for specific related disorders of FTD.

ABCA8 stimulates sphingomyelin production in oligodendrocytes.

Members of the ABCA (ATP-binding cassette subfamily A) family are characterized by their ability to transport lipids across cellular membranes and regulate lipid homoeostasis in the brain and peripheral tissues. ABCA8 is a little-known member of this subfamily that was originally cloned from human brain libraries and has no known function. In an effort to elucidate the role of ABCA8 in the brain we first undertook a comprehensive analysis of its expression in the human brain. ABCA8 was differentially expressed in multiple regions of adult human brains with significantly higher expression in oligodendrocyte-enriched white matter regions compared with grey matter cortical regions. We then assessed the impact of ABCA8 on sphingomyelin production in oligodendrocyte and showed that ABCA8 was able to significantly stimulate both sphingomyelin synthase 1 expression and sphingomyelin production. Furthermore, ABCA8 expression in the prefrontal cortex across the human life span correlated strongly with age-associated myelination, and the myelinating gene p25α was significantly up-regulated with ABCA8. The present study represents the first extensive expression and functional study of ABCA8 in the human brain and the results strongly suggest that ABCA8 regulates lipid metabolism in oligodendrocytes and potentially plays a role in myelin formation and maintenance.

Myriocin-mediated up-regulation of hepatocyte apoA-I synthesis is associated with ERK inhibition.

Sphingolipids including sphingomyelin have been implicated as potential atherogenic lipids. Studies in apoE (apolipoprotein E)-null mice have revealed that the serine palmitoyltransferase inhibitor myriocin reduces plasma levels of sphingomyelin, ceramide, sphingosine-1-phosphate and glycosphingolipids and that this is associated with potent inhibition of atherosclerosis. Interestingly, hepatic apoA-I (apolipoprotein A-I) synthesis and plasma HDL (high-density lipoprotein)-cholesterol levels were also increased in apoE-null mice treated with myriocin. Since myriocin is a known inhibitor of ERK (extracellular-signal-related kinase) phosphorylation, we assessed the possibility that myriocin may be acting to increase hepatic apoA-I production via this pathway. To address this, HepG2 cells and primary mouse hepatocytes were treated with 200 muM myriocin for up to 48 h. Myriocin increased apoA-I mRNA and protein levels by approx. 3- and 2-fold respectively. Myriocin also increased apoA-I secretion up to 3.5-fold and decreased ERK phosphorylation by approx. 70%. Similar findings were obtained when primary hepatocytes were isolated from apoE-null mice that were treated with myriocin (intraperitoneal injection at a dose of 0.3 mg/kg body weight). Further experiments revealed that the MEK (mitogen-activated protein kinase/ERK kinase) inhibitor PD98059 potently inhibited ERK phosphorylation, as expected, and increased primary hepatocyte apoA-I production by 3-fold. These results indicate that ERK phosphorylation plays a role in regulating hepatic apoA-I expression and suggest that the anti-atherogenic mechanism of action for myriocin may be linked to this pathway.

Reduction of plasma glycosphingolipid levels has no impact on atherosclerosis in apolipoprotein E-null mice.

Glycosphingolipids (GSLs) have been implicated as potential atherogenic lipids. Studies in apolipoprotein E-null (apoE(-/-)) mice indicate that exacerbated tissue GSL accumulation resulting from alpha-galactosidase deficiency promotes atherosclerosis, whereas the serine palmitoyl transferase inhibitor myriocin (which reduces plasma and tissue levels of several sphingolipids, including sphingomyelin, ceramide, sphingosine-1-phosphate, and GSLs) inhibits atherosclerosis. It is not clear whether GSL synthesis inhibition per se has an impact on atherosclerosis. To address this issue, apoE(-/-) mice maintained on a high-fat diet were treated with a potent glucosylceramide synthesis inhibitor, d-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4), 10 mg/kg/day for 94 days, and lesion development was compared in mice that were treated with vehicle only. EtDO-P4 reduced plasma GSL concentration by approximately 50% but did not affect cholesterol or triglyceride levels. Assessment of atherosclerotic lesions at four different sites indicated that EtDO-P4 had no significant impact on lesion area. Thus, despite the previously observed positive correlations between plasma and aortic GSL concentrations and the development of atherosclerosis, and the in vitro evidence implying that GSLs may be pro-atherogenic, our current data indicate that inhibition of GSL synthesis does not inhibit atherosclerosis in vivo.

Myriocin slows the progression of established atherosclerotic lesions in apolipoprotein E gene knockout mice.

The serine palmitoyl transferase inhibitor myriocin potently suppresses the development of atherosclerosis in apolipoprotein E (apoE) gene knockout (apoE(-/-)) mice fed a high-fat diet. This is associated with reduced plasma sphingomyelin (SM) and glycosphingolipid levels. Furthermore, oral administration of myriocin decreases plasma cholesterol and triglyceride (TG) levels. Here, we aimed to determine whether myriocin could inhibit the progression (or stimulate the regression) of established atherosclerotic lesions and to examine potential changes in hepatic and plasma lipid concentrations. Adult apoE(-/-) mice were fed a high-fat diet for 30 days, and lesion formation was histologically confirmed. Replicate groups of mice were then transferred to either regular chow or chow containing myriocin (0.3 mg/kg/day) and maintained for a further 60 days. Myriocin significantly inhibited the progression of established atherosclerosis when combined lesion areas (aortic sinus, arch, and celiac branch point) were measured. Although the inhibition of lesion progression was observed mainly in the distal regions of the aorta, regression of lesion size was not detected. The inhibition of lesion progression was associated with reductions in hepatic and plasma SM, cholesterol, and TG levels and increased hepatic and plasma apoA-I levels, indicating that the modulation of pathways associated with several classes of atherogenic lipids may be involved.

Inhibition of atherosclerosis by the serine palmitoyl transferase inhibitor myriocin is associated with reduced plasma glycosphingolipid concentration.

Glycosphingolipids (GSL) have been implicated as potential atherogenic lipids. Inhibition of hepatic serine palmitoyl transferase (SPT) reduces plasma sphingomyelin (SM) levels in the absence of changes in cholesterol or triglyceride (TG) concentration and this leads to a reduction of atherosclerosis in apolipoprotein-E gene knockout (apoE(-/-)) mice. The possibility that the reduced atherosclerosis resulting from SPT inhibition is associated with decreases in plasma GSL concentration has not been examined and was the primary aim of this investigation. We show that intraperitoneal delivery of the SPT inhibitor myriocin for 9 weeks inhibits atherosclerosis in apoE(-/-) mice fed a high fat diet. Lesion inhibition was most pronounced at the aortic arch and distal sites of the thoracic and abdominal aorta. There was also a trend towards a reduction in lesion area at the aortic root. Myriocin treatment resulted in significant reductions in both plasma SM and GSL concentration of 42% and 25%, as assessed by enzymatic and HPLC methods, respectively. Moreover, SM and GSL concentrations were significantly correlated, indicating that SPT inhibition suppresses the synthesis of both these sphingolipids concomitantly. The inhibition of atherosclerosis induced by myriocin was not associated with changes in plasma cholesterol or TG concentrations or lipoprotein profiles as determined by FPLC. These data indicate that therapeutic reduction of plasma SM and/or GSL concentrations may offer a novel treatment for atherosclerosis.

Quantitation of ATP-binding cassette subfamily-A transporter gene expression in primary human brain cells.

Five ATP-binding cassette (ABC) subfamily-A transporters (ABCA1, ABCA2, ABCA3, ABCA7 and ABCA8) are expressed in the brain. These transporters may regulate brain lipid transport; however, their relative expression level in isolated human brain cells is unknown. We developed real-time polymerase chain reaction assays to quantify the expression of these genes in human neurons, astrocytes, oligodendrocytes, microglia and cell lines. Neurons expressed predominantly ABCA1 and ABCA3; astrocytes ABCA1, ABCA2 and ABCA3; microglia ABCA1 and oligodendrocytes ABCA2 and ABCA3. Although ABCA7 and ABCA8 expression was relatively low in all cells, the highest expression occurred in microglia and neurons, respectively. ABCA gene expression in the NTERA-2 and MO3.13 cell lines closely resembled the ABCA expression pattern of primary neurons and oligodendrocytes, respectively.

Phosphorylation of apolipoprotein-E at an atypical protein kinase CK2 PSD/E site in vitro.

Apolipoprotein-E (apoE) plays an important role in neuronal lipid transport and is thought to stabilize microtubules by preventing tau hyperphosphorylation. ApoE is also associated with insoluble amyloid detected in Alzheimer disease brain lesions. The apoE C-terminal shares several physicochemical features with alpha-synuclein, another neuronal apolipoprotein-like protein. Alpha-synuclein is phosphorylated by protein kinase CK2 (CK2) at an atypical PSD/E motif in vivo and in vitro. We identified a similar PSD/E motif in apoE and therefore investigated its potential phosphorylation by CK2 in vitro. When a [(32)P]-labeling approach was used, CK2 readily phosphorylated purified human apoE as well as recombinant forms of human apoE3 and apoE4. Using liquid chromatography mass spectrometry techniques, we mapped the major apoE CK2 phosphorylation site to Ser296 within the apoE PSD/E motif. We also found that apoE potently activated CK2 as demonstrated by increased CK2beta subunit autophosphorylation and by increased phosphorylation of tau when the latter was added to the kinase reaction mixtures. Other proteins such as apolipoprotein A-I and albumin did not effectively activate CK2. The phosphorylation of apoE by CK2 as well as the activation of CK2 by apoE may be relevant in vivo where apoE, CK2, and tau are co-localized with additional CK2 targets on neuronal microtubules.

Glycosphingolipid accumulation inhibits cholesterol efflux via the ABCA1/apolipoprotein A-I pathway: 1-phenyl-2-decanoylamino-3-morpholino-1-propanol is a novel cholesterol efflux accelerator.

Cellular glycosphingolipid (GSL) storage is known to promote cholesterol accumulation. Although physical interactions between GSLs and cholesterol are thought to cause intracellular cholesterol "trapping," it is not known whether cholesterol homeostatic mechanisms are also impaired under these conditions. ApoA-I-mediated cholesterol efflux via ABCA1 (ATP-binding cassette transporter A1) is a key regulator of cellular cholesterol balance. Here, we show that apoA-I-mediated cholesterol efflux was inhibited (by up to 53% over 8 h) when fibroblasts were treated with lactosylceramide or the glucocerebrosidase inhibitor conduritol B epoxide. Furthermore, apoA-I-mediated cholesterol efflux from fibroblasts derived from patients with genetic GSL storage diseases (Fabry disease, Sandhoff disease, and GM1 gangliosidosis) was impaired compared with control cells. Conversely, apoA-I-mediated cholesterol efflux from fibroblasts and cholesterol-loaded macrophage foam cells was dose-dependently stimulated (by up to 6-fold over 8 h) by the GSL synthesis inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Unexpectedly, a structurally unrelated GSL synthesis inhibitor, N-butyldeoxynojirimycin, was unable to stimulate apoA-I-mediated cholesterol efflux despite achieving similar GSL depletion. PDMP was found to up-regulate ABCA1 mRNA and protein expression, thereby identifying a contributing mechanism for the observed acceleration of cholesterol efflux to apoA-I. This study reveals a novel defect in cellular cholesterol homeostasis induced by GSL storage and identifies PDMP as a new agent for enhancing cholesterol efflux via the ABCA1/apoA-I pathway.

Heterogeneous expression of apolipoprotein-E by human macrophages.

Apolipoprotein-E (apoE) is expressed at high levels by macrophages. In addition to its role in lipid transport, macrophage-derived apoE plays an important role in immunoregulation. Previous studies have identified macrophage subpopulations that differ substantially in their ability to synthesize specific cytokines and enzymes, however, potential heterogeneous macrophage apoE expression has not been studied. Here we examined apoE expression in human THP-1 macrophages and monocyte-derived macrophages (MDM). Using immunocytochemistry and flow cytometry methods we reveal a striking heterogeneity in macrophage apoE expression in both cell types. In phorbol-ester-differentiated THP-1 macrophages, 5% of the cells over-expressed apoE at levels more than 50-fold higher than the rest of the population. ApoE over-expressing THP-1 macrophages contained condensed/fragmented nuclei and increased levels of activated caspase-3 indicating induction of apoptosis. In MDM, 3-5% of the cells also highly over-expressed apoE, up to 50-fold higher than the rest of the population; however, this was not associated with obvious nuclear alterations. The apoE over-expressing MDM were larger, more granular, and more autofluorescent than the majority of cells and they contained numerous vesicle-like structures that appeared to be coated by apoE. Flow cytometry experiments indicated that the apoE over-expressing subpopulation of MDM were positive for CD14, CD11b/Mac-1 and CD68. These observations suggest that specific macrophage subpopulations may be important for apoE-mediated immunoregulation and clearly indicate that subpopulation heterogeneity should be taken into account when investigating macrophage apoE expression.

Immune response to enzyme replacement therapy: single epitope control of antigen distribution from circulation.

Immune response to replacement therapy has been reported for a range of therapeutic strategies being developed for the treatment of patients with genetic disease. The potential problem of immune response to enzyme replacement therapy has been investigated in alpha-L-iduronidase immunized rats, representing a model of the lysosomal storage disorder Hurler syndrome (alpha-L-iduronidase deficiency). The antibody response to alpha-L-iduronidase showed that the positional location of antibody reactivity was similar for different immunized rats, but the precise linear sequence epitopes identified, varied between rats. A monoclonal antibody reacting to an epitope in close proximity to one high antigenicity site on alpha-L-iduronidase was used to reproduce the in vivo effect of altered enzyme tissue distribution, previously observed in immunized rats infused with alpha-L-iduronidase. The study demonstrated that during an immune response, antibody reacting to a single epitope could partially control the tissue distribution of antigen from circulation.