Thiazolidinediones (TZDs) improve insulin resistance by activating a nuclear hormone receptor, peroxisome proliferator-activated receptor γ (PPARγ). However, the use of TZDs is associated with plasma volume expansion through a mechanism that remains to be clarified. Here we showed that TZDs rapidly stimulate sodium-coupled bicarbonate absorption from the renal proximal tubule in vitro and in vivo. TZD-induced transport stimulation is dependent on PPARγ-Src-EGFR-ERK and observed in rat, rabbit and human, but not in mouse proximal tubules where Src-EGFR is constitutively activated. The existence of PPARγ-Src-dependent nongenomic signaling, which requires the ligand-binding ability, but not the transcriptional activity of PPARγ, is confirmed in mouse embryonic fibroblast cells. The enhancement of the association between PPARγ and Src by TZDs supports an indispensable role of Src in this signaling. These results suggest that the PPARγ-dependent nongenomic stimulation of renal proximal transport is also involved in TZD-induced volume expansion.
ϩ -HCO 3 Ϫ cotransporter (NBC-1) cause proximal renal tubular acidosis (pRTA) associated with ocular abnormalities. One pRTA patient had increased serum amylase, suggesting possible evidence of pancreatitis. To further delineate a link between NBC-1 inactivation and pancreatic dysfunction, immunohistochemical analysis was performed on rat and human pancreas using antibodies against kidney-type (kNBC-1) and pancreatic-type (pNBC-1) transporters. In rat pancreas, the anti-pNBC-1 antibody labeled acinar cells and both apical and basolateral membranes of medium and large duct cells. In human pancreas, on the other hand, the anti-pNBC-1 antibody did not label acinar cells, although it did label the basolateral membranes of the entire duct system. The labeling by anti-kNBC-1 antibody was detected in only a limited number of rat pancreatic duct cells. To examine the effects of pRTA-related mutations, R342S and R554H, on pNBC-1 function, we performed functional analysis and found that both mutants had reduced transport activities compared with the wild-type pNBC-1. These results indicate that pNBC-1 is the predominant variant that mediates basolateral HCO 3 Ϫ uptake into duct cells in both rat and human pancreas. The loss of pNBC-1 function is predicted to have significant impact on overall ductal HCO 3 Ϫ secretion, which could potentially lead to pancreatic dysfunction. Na ϩ -HCO 3 Ϫ cotransporter; pRTA; pancreatic duct cells; pancreatitis THE NA ϩ -HCO 3 Ϫ COTRANSPORTER (NBC-1), originally cloned from amphibian kidney (32), has multiple functions. In the renal proximal tubules, it mediates HCO 3 Ϫ efflux from cells (7, 36, 42), whereas in other epithelial cells, such as the pancreatic duct cells and the corneal endothelium, it mediates HCO 3 Ϫ influx into cells (22,23, 41). The expression of NBC-1 in nonepithelial cells such as trabecular meshwork cells or neurons has been also demonstrated (35, 40). Up to now, three NBC-1 variants have been identified: kNBC-1 from kidney, pNBC-1 from pancreas, and hNBC-1 from heart. Although kNBC-1 differs from pNBC-1 only at the NH 2 terminus, pNBC-1 is identical to hNBC-1 at the amino acid level and differs only in its 5Ј-untranslated region (1,8,10). These NBC-1 variants originate from the same SLC4A4 gene by alternative splicing (2). It is therefore predictable that mutational inactivation of NBC-1 variants may induce a variety of clinical manifestations, including renal and extrarenal phenotypes. Indeed, we have recently shown that mutations in the common coding region of NBC-1, corresponding to R298S and R510H in kNBC-1 or R342S and R554H in pNBC-1, cause renal proximal tubular acidosis (pRTA) associated with ocular abnormalities such as band keratopathy, glaucoma, and cataracts (19). The expression study in ECV304 cells showed that the kidneytype mutants, R298S and R510H, have reduced transport activities compared with the wild-type kNBC-1 (19). The expression of both kNBC-1 and pNBC-1 in several ocular tissues has been confirmed (6, 40). It remains to be clarified, however, w...
Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor that mediates the selective uptake of HDL cholesterol and cholesterol secretion into bile in the liver. Previously, we identified an SR-BI-associated protein, termed PDZK1, from rat liver membrane extracts. PDZK1 contains four PSD-95͞ Dlg͞ZO-1 (PDZ) domains, the first of which in the N-terminal region is responsible for the association with SR-BI. PDZK1 controls hepatic SR-BI expression in a posttranscriptional fashion both in cell culture and in vivo. In this study, we demonstrated that the C-terminal region of PDZK1 is crucial for up-regulating SR-BI protein expression. Metabolic labeling experiments and phosphoamino acid analysis revealed that PDZK1 is phosphorylated at Ser residues within this region. Point-mutation analysis demonstrated that PDZK1 is phosphorylated at Ser-509. Interestingly, a mutant PDZK1, in which Ser-509 was replaced with Ala, lost the ability to up-regulate SR-BI protein. We identified Ser-509 of PDZK1 as the residue that is phosphorylated by the cAMP-dependent PKA in vitro as well as in cell culture. Ser-509 of PDZK1 in rat liver was also phosphorylated, as shown by an Ab that specifically detects phosphorylated Ser-509. Administration of glucagon to Wistar rats increased PDZK1 phosphorylation as well as hepatic SR-BI and PDZK1 expression while it decreased plasma HDL levels, indicating that PDZK1 phosphorylation is hormonally regulated. These findings suggest that phosphorylation of PDZK1 has an important role in the regulation of hepatic SR-BI expression and, thus, influences plasma HDL levels.glucagon ͉ high-density lipoprotein ͉ PKA
Objective-Plasma platelet-activating factor (PAF) acetylhydrolase (AH) is an enzyme bound with lipoproteins that degrades not only PAF but also PAF-like oxidized phospholipids that are proposed to promote atherosclerosis. In this study, we investigated the distribution of PAF-AH protein among lipoprotein classes by using adenovirus-mediated gene transfer in mice, and we examined its effects on lipoprotein oxidation and foam cell formation of macrophages. Methods and Results-Adenovirus-mediated overexpression of PAF-AH in mice resulted in a 76-to 140-fold increase in plasma PAF-AH activity. Contrary to the previous report, overexpressed human PAF-AH protein was bound to very low density lipoprotein, intermediate density lipoprotein, low density lipoprotein, and high density lipoprotein (HDL). All the lipoproteins with overexpressed human PAF-AH revealed more resistance against oxidative stress, which was associated with lower levels in autoantibody against oxidized low density lipoprotein in the plasma. In addition, HDL with human PAF-AH inhibited foam cell formation and facilitated cholesterol efflux in macrophages. Key Words: platelet-activating factor acetylhydrolase Ⅲ oxidative stress Ⅲ adenovirus Ⅲ foam cell formation Ⅲ cholesterol efflux P latelet-activating factor (PAF) acetylhydrolase (AH) is a calcium-independent enzyme that degrades PAF, a bioactive phospholipid mediator for allergic and inflammatory processes, to a biologically inactive lyso-PAF. Plasma PAF-AH, 1 of the 3 PAF-AH isoforms identified so far, is produced from macrophages and exists in the plasma in the form bound with lipoproteins; the other 2 isoforms are found only in tissues. Seventy percent to 83% of the plasma PAF-AH protein exists on LDL, and 11% to 30% exists on HDL in human plasma. 1,2 An interchange between the 2 lipoproteins has been reported in plasma PAF-AH. 1 In mice, it has been recognized that PAF-AH is associated primarily with HDL and minimally with VLDL 3,4 and that neither murine PAF-AH nor human PAF-AH has been proposed to bind to murine LDL. 5 An observational study has shown that plasma PAF-AH activity is altered in atherosclerotic diseases. 6 Oxidation of LDL, in which PAF-like oxidized phospholipids are produced on the LDL surface, is one of the key factors in the early stages of atherosclerosis. 7 Besides catalyzing PAF, plasma PAF-AH protein hydrolyzes PAF-like oxidized phospholipids, thereby most likely inactivating the biologically active mediator. However, the products of this reaction include oxidized fatty acids and lysophosphatidylcholine, 8 which are potentially inflammatory mediators that could amplify atherogenesis. Therefore, it is not fully clear whether PAF-AH is antiatherogenic or proatherogenic in humans. There was one report documenting that high PAF-AH activity is associated with an increased risk of coronary artery disease in humans. 6 However, it is not conclusive whether PAF-AH is a causative agent of coronary artery disease or just a marker. A recent animal study demonstrated that overexpressi...
Cholesterol ester (CE)-laden foam cells are a hallmark of atherosclerosis. To determine whether stimulation of the hydrolysis of cytosolic CE can be used as a novel therapeutic modality of atherosclerosis, we overexpressed hormone-sensitive lipase (HSL) in THP-1 macrophage-like cells by adenovirus-mediated gene delivery, and we examined its effects on the cellular cholesterol trafficking. We show here that the overexpression of HSL robustly increased neutral CE hydrolase activity and completely eliminated CE in the cells that had been preloaded with CE by incubation with acetylated low density lipoprotein. In these cells, cholesterol efflux was stimulated in the absence or presence of high density lipoproteins, which might be at least partially explained by the increase in the expression of ABCA1. Importantly, these effects were achieved without the addition of acyl-CoA:cholesterol acyltransferase inhibitor, cAMP, or even high density lipoproteins. Furthermore, the uptake and degradation of acetylated low density lipoprotein was significantly reduced probably by decreased expression of scavenger receptor A and CD36. Notably, the cells with stimulated CE hydrolysis did not exhibit either buildup of free cholesterol or cytotoxicity. In conclusion, increased hydrolysis of CE by the overexpression of HSL leads to complete elimination of CE from THP-1 foam cells not only by increasing efflux but also by decreasing influx of cholesterol. Cholesterol ester (CE)1 -laden macrophage foam cells are a hallmark of fatty streak lesions in atherosclerotic plaques.Besides cleaning up extracellularly deposited atherogenic lipoproteins, macrophage foam cells secrete a wide variety of substances, which include inflammatory cytokines, chemokines, growth factors, and proteases (1). Furthermore, lipidrich lesions that are characterized by a plethora of macrophage foam cells are associated with massive infiltration with activated T lymphocytes and prone to rupture, thereby leading to thrombotic coronary occlusion (2). Thus, elimination of CE from foam cells is potentially a promising therapeutic strategy to stabilize rupture-prone atherosclerotic plaques.Foam cells are generated by the uptake of modified lipoproteins through scavenger receptors, such as scavenger receptor A (SR-A) and CD36 (3). Hydrolysis of the lipoprotein-associated CE by lysosomal acid lipase liberates free cholesterol (FC), which is subsequently re-esterified by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) to form CE for storage in the cytoplasmic lipid droplets (4, 5). The cytoplasmic CE is in turn hydrolyzed by neutral CE hydrolase (NCEH) to generate FC, which is transported to a compartment for re-esterification by ACAT1. The rest of the FC is released out of the cells primarily through ATP-binding cassette transporter A1 (ABCA1) (6, 7). Thus, the balance between synthesis and hydrolysis of CE conceivably governs the level of CE in macrophages.Inhibition of ACAT activity is shown to suppress CE accumulation with concomitant promotion of the net hydrolysis of ...
Objective-Apolipoprotein E (apoE) mediates cellular cholesterol efflux and plays a crucial role in the inhibition of atherogenesis. We investigated whether there is an isoform-specific difference in its function for cholesterol efflux from cholesterol-loaded RAW264.7 cells, a murine macrophage cell line that lacks endogenous apoE expression. Methods and Results-When human apoE was expressed in RAW264.7 cells, apoE2 reduced cellular total cholesterol (TC) and esterified cholesterol (EC) levels significantly, whereas apoE3 and apoE4 had no effect. However, treatment of cells with 4-methylumbelliferyl-7--D-xyloside (-DX) resulted in all 3 isoforms' reducing cellular TC and EC contents significantly. We also investigated the effect of exogenously derived apoE on cholesterol efflux by utilizing the medium harvested from HeLa cells expressing apoE. ApoE2 and E3 reduced both cellular TC and EC contents significantly, whereas apoE4 did not. However, treatment of the cells with -DX resulted in all 3 exogenously derived apoE isoforms' reducing TC and EC contents significantly. The binding ability of apoE to heparan sulfate proteoglycans examined by heparinase I treatment revealed less binding ability of apoE2 compared with that of apoE3 or apoE4. Conclusions-The present study clarified the differential cellular cholesterol-modulating effect of apoE isoforms in macrophages, which would be due to the difference in their binding to proteoglycans.
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