Contribution of adipocyte Na/K-ATPase α1/CD36 signaling induced exosome secretion in response to oxidized LDL

IntroductionAdipose tissue constantly secretes adipokines and extracellular vesicles including exosomes to crosstalk with distinct tissues and organs for whole-body homeostasis. However, dysfunctional adipose tissue under chronic inflammatory conditions such as obesity, atherosclerosis, and diabetes shows pro-inflammatory phenotypes accompanied by oxidative stress and abnormal secretion. Nevertheless, molecular mechanisms of how adipocytes are stimulated to secrete exosomes under those conditions remain poorly understood.MethodsMouse and human in vitro cell culture models were used for performing various cellular and molecular studies on adipocytes and macrophages. Statistical analysis was performed using Student's t-test (two-tailed, unpaired, and equal variance) for comparisons between two groups or ANOVA followed by Bonferroni's multiple comparison test for comparison among more than two groups.Results and discussionIn this work, we report that CD36, a scavenger receptor for oxidized LDL, formed a signaling complex with another membrane signal transducer Na/K-ATPase in adipocytes. The atherogenic oxidized LDL induced a pro-inflammatory response in in vitro differentiated mouse and human adipocytes and also stimulated the cells to secrete more exosomes. This was largely blocked by either CD36 knockdown using siRNA or pNaKtide, a peptide inhibitor of Na/K-ATPase signaling. These results showed a critical role of the CD36/Na/K-ATPase signaling complex in oxidized LDL-induced adipocyte exosome secretion. Moreover, by co-incubation of adipocyte-derived exosomes with macrophages, we demonstrated that oxidized LDL-induced adipocyte-derived exosomes promoted pro-atherogenic phenotypes in macrophages, including CD36 upregulation, IL-6 secretion, metabolic switch to glycolysis, and mitochondrial ROS production. Altogether, we show here a novel mechanism through which adipocytes increase exosome secretion in response to oxidized LDL and that the secreted exosomes can crosstalk with macrophages, which may contribute to atherogenesis

Modification of HDL by reactive aldehydes alters select cardioprotective functions of HDL in macrophages

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154382/1/febs15034_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154382/2/febs15034.pd

Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by Enhancing Scavenger Receptor Class B1 (SR-BI)-Mediated High-Density Lipoprotein (HDL)-Cholesteryl Ester Uptake

Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr-/-, PCPE2-/- mice, which had elevated HDL levels compared with LDLr-/- mice with similar LDL concentrations. We found that LDLr-/-, PCPE2-/- mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr-/- mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr-/-, PCPE2-/- mice was similar to that reported for LDLr-/-, apoA-I-/- mice, which lack any apoA-I/HDL. Furthermore, LDLr-/-, PCPE2-/- mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr-/- mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system

Cholesterol and Lipoprotein Dynamics in a Hibernating Mammal

Hibernating mammals cease feeding during the winter and rely primarily on stored lipids to fuel alternating periods of torpor and arousal. How hibernators manage large fluxes of lipids and sterols over the annual hibernation cycle is poorly understood. The aim of this study was to investigate lipid and cholesterol transport and storage in ground squirrels studied in spring, summer, and several hibernation states. Cholesterol levels in total plasma, HDL and LDL particles were elevated in hibernators compared with spring or summer squirrels. Hibernation increased plasma apolipoprotein A-I expression and HDL particle size. Expression of cholesterol 7 alpha-hydroxylase was 13-fold lower in hibernators than in active season squirrels. Plasma triglycerides were reduced by fasting in spring but not summer squirrels. In hibernators plasma β-hydroxybutyrate was elevated during torpor whereas triglycerides were low relative to normothermic states. We conclude that the switch to a lipid-based metabolism during winter, coupled with reduced capacity to excrete cholesterol creates a closed system in which efficient use of lipoproteins is essential for survival

Functional characterization of newly-discovered mutations in human SR-BI.

In rodents, SR-BI has been firmly established as a physiologically relevant HDL receptor that mediates removal of HDL-cholesteryl esters (CE). However, its role in human lipoprotein metabolism is less defined. Recently, two unique point mutations in human SR-BI - S112F or T175A - were identified in subjects with high HDL-cholesterol (HDL-C) levels. We hypothesized that mutation of these conserved residues would compromise the cholesterol-transport functions of SR-BI. To test this hypothesis, S112F- and T175A-SR-BI were generated by site-directed mutagenesis. Cell surface expression was confirmed for both mutant receptors in COS-7 cells upon transient transfection, albeit at lower levels for T175A-SR-BI. Both mutant receptors displayed defective HDL binding, selective uptake of HDL-CE and release of free cholesterol (FC) from cells to HDL. Mutant receptors were also unable to re-organize plasma membrane pools of FC. While these impaired functions were independent of receptor oligomerization, inability of T175A-SR-BI to mediate cholesterol-transport functions could be related to altered N-linked glycosylation status. In conclusion, high HDL-C levels observed in carriers of S112F- or T175A-SR-BI mutant receptors are consistent with the inability of these SR-BI receptors to mediate efficient selective uptake of HDL-CE, and suggest that increased plasma HDL concentrations in these settings may not be associated with lower risk of cardiovascular disease

Cell surface expression of wild-type and mutant SR-BI receptors.

<p>(A) COS-7 cells expressing wild-type or mutant SR-BI receptors were assessed for cell surface expression following incubation with NHS-LC-Biotin as described in Materials and Methods. Immunoblot analyses of biotinylated SR-BI at the cell surface (from ∼150 µg of total lysate) (top panel) and in 20 µg of total cell lysate (middle panel) are shown using an antibody directed against the C-terminal cytoplasmic domain of SR-BI. GAPDH was detected as a loading control (bottom panel). The numbers above the top panel represent cell surface receptor expression by densitometry analysis (where SR-BI = 100%). Data are representative of 3 independent experiments. (B) Surface expression of wild-type or mutant SR-BI receptors in COS-7 cells was assessed by flow cytometry using an antibody directed against the extracellular domain of SR-BI. Data are expressed as a % of SR-BI expression following subtraction of empty vector values. Data are the average of 12 independent transfections.</p

Mutant SR-BI receptors are unable to mediate efflux of free cholesterol from cells to HDL.

<p>COS-7 cells transiently expressing wild-type or mutant SR-BI and pre-labeled with [³H]-cholesterol were incubated with HDL (50 µg/mL) for four hours. Cell and media radioactivity were assessed. Combined data from four independent experiments, each performed in quadruplicate, are shown. *p<0.001, as determined by one-way ANOVA.</p

Mutant receptors maintain their ability to form homo-oligomers.

<p>COS-7 cell lysates transiently expressing wild-type or mutant SR-BI were separated by 6% PFO-PAGE. SR-BI was detected by immunoblot analysis using an antibody directed against the C-terminal domain of the receptor. Data is representative of 4 independent experiments.</p

Mutant SR-BI receptors display decreased HDL binding and selective uptake of HDL-COE.

<p>COS-7 cells transiently expressing wild-type or mutant SR-BI were incubated with [¹²⁵I]/[³H]-COE-labeled HDL (10 µg HDL protein/mL). (A) Binding of [¹²⁵I]HDL and (B) selective uptake of [³H]COE are shown. (C, D) Individual data sets from panels A and B were first normalized to wild-type SR-BI (normalized value = 100%) following subtraction of empty vector values. Next, the corrected HDL binding and HDL-COE values (panels C and D, respectively) were calculated by dividing the normalized values for each receptor by the corresponding value for surface expression (by flow cytometry) from parallel wells within the same experiment. Combined data from six independent experiments, each performed in triplicate, are shown. *p<0.001, as determined by one-way ANOVA.</p