Amyloid ␤ protein (A␤) elicits a toxic effect on neurons in vitro and in vivo. In present study we attempt to elucidate the mechanism by which A␤ confers its neurotoxicity. The neuroprotective effects of phytoestrogens on A␤-mediated toxicity were also investigated. Cortical neurons treated with 5 M A␤-(25-35) for 40 h decreased the cell viability by 45.5 ؎ 4.6% concomitant with the appearance of apoptotic morphology. 50 M kaempferol and apigenin decreased the A␤-induced cell death by 81.5 ؎ 9.4% and 49.2 ؎ 9.9%, respectively. A␤ increased the activity of caspase 3 by 10.6-fold and to a lesser extent for caspase 2, 8, and 9. The A␤-induced activation of caspase 3 and release of cytochrome c showed a biphasic pattern. Apigenin abrogated A␤-induced cytochrome c release, and the activation of caspase cascade. Kaempferol showed a similar effect but to a less extent. Kaempferol was also capable of eliminating A␤-induced accumulation of reactive oxygen species. These two events accounted for the remarkable effect of kaempferol on neuroprotection. Quercetin and probucol did not affect the A␤-mediated neurotoxicity. However, they potentiated the protective effect of apigenin. Therefore, these results demonstrate that A␤ elicited activation of caspase cascades and reactive oxygen species accumulation, thereby causing neuronal death. The blockade of caspase activation conferred the major neuroprotective effect of phytoestrogens. The antioxidative activity of phytoestrogens also modulated their neuroprotective effects on A␤-mediated toxicity.

Incubation of 3T3-L1 adipocytes with C2- and C6-ceramides (N-acetyl- and N-hexanoylsphingosines) but not dihydro-C2-ceramide increased 2-deoxyglucose uptake in the absence of insulin. This effect was inhibited by PD 98059, LY 294002, and rapamycin, which block the activation of mitogen-activated protein kinase, phosphatidylinositol (PI) 3-kinase, and ribosomal S6 kinase, respectively. Long-term increases in PI 3-kinase activity associated with insulin receptor substrate 1 (IRS-1) increased GLUT1 and GLUT4 concentrations in plasma membranes. This together with increased GLUT1 (but not GLUT4) synthesis explains the increase in non-insulin-dependent glucose uptake. C2-ceramide inhibited insulin-stimulated glucose uptake after 2 h by decreasing insulin-induced translocation of GLUT1 and GLUT4 to plasma membranes. This occurred when there was no increase in basal glucose uptake or decrease in activation of IRS-1 or PI 3-kinase. Incubation for 24 h with tumor necrosis factor-alpha (TNF-alpha) but not C2-ceramide decreased the concentration and insulin-induced tyrosine phosphorylation of IRS-1 in this experimental system. Cell-permeable ceramides mimic some effects of TNF-alpha, especially in stimulating basal glucose uptake. We identified a site for inhibiting insulin-stimulated glucose uptake that is downstream of PI 3-kinase. Our work provides further mechanisms for the effects of TNF-alpha and ceramides in increasing non-insulin-dependent glucose uptake and decreasing insulin-stimulated uptake in vivo.

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The site of apolipoprotein B (apoB) degradation was investigated in cultured rat hepatocytes. Brefeldin A plus nocodazole completely blocked apoB degradation suggesting the involvement of a post-endoplasmic reticulum (ER) compartment. Monensin inhibited apoB degradation by 40% implying that a post-Golgi compartment could be involved in degradation of apoB. Ammonium chloride or chloroquine inhibited partially the degradation of apoB100 and apoB48, indicating some degradation in lysosomes, or in an acidic compartment such as trans-Golgi or endosomes. The degradations of apoB100 and apoB48 were blocked completely by (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (EST) during a chase of 90 min demonstrating that a cysteine protease was responsible for apoB degradation. Chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, and aprotinin had no significant effect on the degradation of apoB48. However, leupeptin and pepstatin decreased the degradation of apoB100 by 20 -30%. Degradation of apoB100 and apoB48 occurred in isolated Golgi fractions with little degradation in heavy or light ER. Degradation of apoB in Golgi fractions was inhibited by EST and by preincubating hepatocytes with 10 nM dexamethasone. Immunofluorescent microscopy revealed that apoB accumulated in the Golgi region after EST treatment. It is concluded that a major part of apoB degradation in rat hepatocytes occurs in a post-ER compartment via the action of a cysteine protease that is regulated by glucocorticoids.Apolipoprotein B (apoB) plays a central role in the assembly, secretion, and metabolism of triacylglycerol-rich lipoproteins (chylomicrons and VLDL) 1 and LDL (1). There are two forms of apoB in mammals: the larger molecular weight form, apoB100, consists of 4536 amino acids, whereas the smaller form, apoB48, is the amino-terminal 48% of apoB100. Both apoB100 and apoB48 are products of the same gene. ApoB48 mRNA is produced from apoB100 mRNA mainly in the intestine by RNA editing which involves a cytidine deaminase (2-5). Although most mammalian livers produce only apoB100, rat liver synthesizes both apoB100 and apoB48. ApoB is synthesized on polyribosomes bound to the cytoplasmic surface of the ER and then translocates into ER lumen. ApoB translocation has been suggested to involve specific multiple pause-transfer sequences that temporarily arrest the translocation process (6 -8).Changes in the lipid composition in microsomal membranes also diminish apoB translocation across ER membranes (9). Several studies suggested that association of apoB with the full complement of lipids occurs in ER (10 -13). Some experiments, however, indicated that the majority of triacylglycerols and phospholipids are assembled into VLDL particles in the Golgi (14 -16).Pulse-chase studies suggest that a significant proportion of the apoB synthesized de novo in rat hepatocytes is degraded intracellularly (17-19). Intracellular degradation of apoB has also been observed in HepG2 cells (20 -22), and the degradation may be important in...

Oversecretion of apoB and decreased removal of apoB-containing lipoproteins by the liver results in hyperapobetalipoproteinemia, which is a risk factor for atherosclerosis. We investigated how dexamethasone, a synthetic glucocorticoid, affects the synthesis, degradation, and secretion of apoB-100 and apoB-48. Primary rat hepatocytes were incubated with dexamethasone for 16 hours. Incorporation of [35S]methionine into apoB-48 and apoB-100 was increased by 36% and 50%, respectively, with 10 nmol/L dexamethasone, despite a 28% decrease of incorporation into total cell proteins. However, Northern blot analysis revealed that dexamethasone (1 to 1000 nmol/L) did not significantly alter the steady-state concentrations of apoB mRNA, suggesting that the net increase in apoB synthesis may involve increased translational efficiency. The intracellular retention and the rate and efficiency of apoB secretion were determined by pulse-chase experiments in which the hepatocytes were labeled with [35S]methionine for 10 minutes or 1 hour, and the disappearance of labeled apoB from the cells and its accumulation in the medium were monitored. Degradation of labeled apoB-100 after a 3-hour chase in both protocols was decreased from about 50% to 30%, whereas degradation of apoB-48 was decreased from 30% to 10% to 20% by treatment with 10 or 100 nmol/L dexamethasone. Additionally, the half-life of decay (time required for 50% of labeled cell apoB-100 to disappear from the peak of radioactivity following a 10-minute pulse) was increased by treatment with 10 nmol/L dexamethasone from 77 to 112 minutes, and the value for apoB-48 increased from 145 to 250 minutes. Treatment with 100 nmol/L dexamethasone also stimulated secretion of 35S-labeled apoB-100 and apoB-48 by twofold and 1.5-fold, respectively. The increased secretion of apoB-100 and apoB-48 after dexamethasone treatment was confirmed by immunoblot analysis for apoB mass, and the effect was relatively specific since albumin secretion was not significantly changed. We conclude that glucocorticoids promote the secretion of hepatic apoB-containing lipoproteins by increasing the net synthesis of apoB-100 and apoB-48 and by decreasing the intracellular degradation of newly synthesized apoB. An increased action of glucocorticoids coupled with a decreased ability of insulin to suppress these effects in insulin resistance can lead to hyperapobetalipoproteinemia and an increased risk of atherosclerosis.