Asymmetrical regulation of scavenger receptor class B type I by apical and basolateral stimuli using Caco-2 cells

Cholesterol uptake and the mechanisms that regulate cholesterol translocation from the intestinal lumen into enterocytes remain for the most part unclear. Since scavenger receptor class B type I (SR-BI) has been suggested to play a role in cholesterol absorption, we investigated cellular SR-BI modulation by various potential effectors administered in both apical and basolateral sides of Caco-2 cells. With differentiation, Caco-2 cells increased SR-BI protein expression. Western blot analysis showed the ability of cholesterol and oxysterols in both cell compartments to reduce SR-BI protein expression. Among the n-3, n-6, and n-9 fatty acid families, only eicosapentaenoic acid was able to lower SR-BI protein expression on both sides, whereas apical alpha-linolenic acid decreased SR-BI abundance and basolateral arachidonic acid (AA) raised it. Epidermal growth factor and growth hormone, either in the apical or basolateral medium, diminished SR-BI cellular content, while insulin displayed the same effect only on the basolateral side. In the presence of proinflammatory agents (LPS, TNF-alpha, IFN-gamma), Caco-2 cells exhibited differential behavior. SR-BI was downregulated by lipopolysaccharide on both sides. Finally, WY-14643 fibrate diminished SR-BI protein expression when it was added to the apical medium. Biotinylation studies in response to selected stimuli revealed that regulatory modifications in SR-BI protein expression occurred for the most part at the apical cell surface irrespective of the effector location. Our data indicate that various effectors supplied to the apical and basolateral compartments may impact on SR-BI at the apical membrane, thus suggesting potential regulation of intestinal cholesterol absorption and distribution in various intracellular pools.     

FIC1, a P-type ATPase linked to cholestatic liver disease,has homologues (ATP8B2 and ATP8B3) expressed throughout the body

ATP8B1/FIC1 is a member of the Type IV P-type ATPase family, which function as ATP dependent aminophospholipid translocases (APLT). We identified two familial intrahepatic cholestasis type 1 (FIC1) homologues, ATP8B2 and ATP8B3, with 53% and 45% amino acid identity, respectively. The expression profile for each gene was determined using a 73-tissue human RNA expression array. The subfamily of FIC1-like proteins is expressed in a wide range of tissues. Given that mutations in FIC1 result in liver disease, these proteins may have important roles in other organs in which they are candidates for genetic and acquired diseases.     

Phospholipase D1 decreases type I collagen levels in hepatic stellate cells via induction of autophagy

Hepatic stellate cells (HSCs) are major players in liver fibrogenesis. Accumulating evidence shows that suppression of autophagy plays an important role in the development and progression of liver disease. Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA) and choline, was recently shown to modulate autophagy. However, little is known about the effects of PLD1 on the production of type I collagen that characterizes liver fibrosis. Here, we examined whether PLD1 regulates type I collagen levels in HSCs through induction of autophagy. Adenovirus-mediated overexpression of PLD-1 (Ad-PLD1) reduced type I collagen levels in the activated human HSC lines, hTERT and LX2. Overexpression of PLD1 in HSCs led to induction of autophagy as demonstrated by increased LC3-II conversion and formation of LC3 puncta, and decreased p62 abundance. Moreover, inhibiting the induction of autophagy by treating cells with bafilomycin or a small interfering (si)RNA for ATG7 rescued Ad-PLD1-induced suppression of type I collagen accumulation in HSCs. The effects of PLD on type I collagen levels were not related to TGF-β/Smad signaling. Furthermore, treatment of cells with PA induced autophagy and inhibited type I collagen accumulation. The present study indicates that PLD1 plays a role in regulating type I collagen accumulation through induction of autophagy.     

SphK1 mediates hepatic inflammation in a mouse model of NASH induced by high saturated fat feeding and initiates proinflammatory signaling in hepatocytes

Steatohepatitis occurs in up to 20% of patients with fatty liver disease and leads to its primary disease outcomes, including fibrosis, cirrhosis, and increased risk of hepatocellular carcinoma. Mechanisms that mediate this inflammation are of major interest. We previously showed that overload of saturated fatty acids, such as that which occurs with metabolic syndrome, induced sphingosine kinase 1 (SphK1), an enzyme that generates sphingosine-1-phosphate (S1P). While data suggest beneficial roles for S1P in some contexts, we hypothesized that it may promote hepatic inflammation in the context of obesity. Consistent with this, we observed 2-fold elevation of this enzyme in livers from humans with nonalcoholic fatty liver disease and also in mice with high saturated fat feeding, which recapitulated the human disease. Mice exhibited activation of NFκB, elevated cytokine production, and immune cell infiltration. Importantly, SphK1-null mice were protected from these outcomes. Studies in cultured cells demonstrated saturated fatty acid induction of SphK1 message, protein, and activity, and also a requirement of the enzyme for NFκB signaling and increased mRNA encoding TNFα and MCP1. Moreover, saturated fat-induced NFκB signaling and elevation of TNFα and MCP1 mRNA in HepG2 cells was blocked by targeted knockdown of S1P receptor 1, supporting a role for this lipid signaling pathway in inflammation in nonalcoholic fatty liver disease.     

Effect of vitamin D3 supplementation on hepatic lipid dysregulation associated with autophagy regulatory AMPK/Akt-mTOR signaling in type 2 diabetic mice

Vitamin D₃ has been reported to protect liver against non-alcoholic fatty liver disease (NAFLD) by attenuating hepatic lipid dysregulation in type 2 diabetes mellitus (T2DM). However, the mechanism of vitamin D₃ on hepatic lipid metabolism-associated autophagy in hyperglycemia-induced NAFLD remains yet to be exactly elucidated. C57BL/6J mice were intraperitoneally injected with 30 mg/kg of streptozotocin and fed a high-fat diet for induction of diabetes. All mice were administered with vehicle or vitamin D₃ (300 ng/kg or 600 ng/kg) by oral gavage for 12 weeks. Histological demonstrations of the hepatic tissues were obtained by H&E staining and the protein levels related to lipid metabolism and autophagy signaling were analyzed by Western blot. Treatment with vitamin D₃ improved insulin resistance, liver damage, and plasma lipid profiles, and decreased hepatic lipid content in the diabetic mice. Moreover, vitamin D₃ administration ameliorated hepatic lipid dysregulation by downregulating lipogenesis and upregulating lipid oxidation under diabetic condition. Importantly, vitamin D₃ treatment induced autophagy by activating AMP-activated protein kinase (AMPK), inactivating Akt and ultimately blocking mammalian target of rapamycin (mTOR) activation in the T2DM mice. Additionally, vitamin D₃ was found to be effective in anti-apoptosis and anti-fibrosis in the liver of diabetic mice. The results suggested that vitamin D₃ may ameliorate hepatic lipid dysregulation by activating autophagy regulatory AMPK/Akt-mTOR signaling in T2DM, providing insights into its beneficial effects on NAFLD in type 2 diabetic patients.     

Hepatic cytochrome P450 8B1 and cholic acid potentiate intestinal epithelial injury in colitis by suppressing intestinal stem cell renewal

1. Download : Download high-res image (164KB)
2. Download : Download full-size image

     

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.     

Inhibition of mTOR down-regulates scavenger receptor,class B,type I (SR-BI) expression,reduces endothelial cell migration and impairs nitric oxide production

The mammalian target of rapamycin (mTOR) inhibiting drug rapamycin (Sirolimus) has severe side effects in patients including hyperlipidemia, an established risk factor for atherosclerosis. Recently, it was shown that rapamycin decreases hepatic LDL receptor (LDL-R) expression, which likely contributes to hypercholesterolemia. Scavenger receptor, class B, type I (SR-BI) is the major HDL receptor and consequently regulating HDL-cholesterol levels and the athero-protective effects of HDL. By using the mTOR inhibitor rapamycin, we show that SR-BI is down-regulated in human umbilical vein endothelial cells (HUVECs). This reduction of SR-BI protein as well as mRNA levels by about 50% did not alter HDL particle uptake or HDL-derived lipid transfer. However, rapamycin reduced HDL-induced activation of eNOS and stimulation of endothelial cell migration. The effects on cell migration could be counteracted by SR-BI overexpression, indicating that decreased SR-BI expression is in part responsible for the rapamycin-induced effects. We demonstrate that inhibition of mTOR leads to endothelial cell dysfunction and decreased SR-BI expression, which may contribute to atherogenesis during rapamycin treatment.     

Exposure to novel environment is characterized by an interaction of D1/NMDA receptors underlined by phosphorylation of the NMDA and AMPA receptor subunits and activation of ERK1/2 signaling, leading to epigenetic changes and gene expression in rat hippocampus

Interactions between dopamine and glutamate receptors are essential for prefrontal cortical (PFC) and hippocampal cognitive functions. The hippocampus has been identified as a detector of a novel stimulus, where an association between incoming information and stored memories takes place. Further to our previous results which showed a strong synergistic interaction of dopamine D1 and glutamate NMDA receptors, the present study is going to investigate the functional status of that interaction in rats, following their exposure to a novel environment. Our results showed that the “spatial” novelty induced in rat hippocampus and PFC (a) a significant increase in phosphorylation of NMDA and AMPA receptor subunits, as well as a robust phosphorylation/activation of ERK1/2 signaling, which are both dependent on the concomitant stimulation of D1/NMDA receptors and are both abolished by habituation procedure, (b) chromatin remodeling events (phosphorylation-acetylation of histone H3) and (c) an increase in the immediate early genes (IEGs) c-Fos and zif-268 expression in the CA1 region of hippocampus, which is dependent on the co-activation of D1/NMDA and acetylcholine muscarinic receptors. In conclusion, our results clearly show that a strong synergistic interaction of D1/NMDA receptor is required for the novelty-induced phosphorylation of NMDA and AMPA receptor subunits and for the robust activation of ERK1/2 signaling, leading to chromatin remodeling events and the expression of the IEGs c-Fos and zif-268, which are involved in the regulation of synaptic plasticity and memory consolidation.