Nicotinic acid induces apolipoprotein A-I gene expression in HepG2 and Caco-2 cell lines.

The objective was to test the effect of nicotinic acid on apolipoprotein A-I (apo A-I) gene expression in hepatic (HepG2) and intestinal (Caco-2) cell lines. HepG2 and Caco-2 cells were treated with 0.1, 0.3, 1.0, 3.0, and 10 mmol/L of nicotinic acid; and apo A-I concentrations in conditioned media were measured with Western blots. Relative apo A-I messenger RNA (mRNA) levels, normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA, were measured with quantitative real-time polymerase chain reaction method. The nicotinic acid response element in the apo A-I promoter was identified using a series of apo A-I reporter plasmids containing deletion constructs of the promoter. In other experiments, HepG2 cells were also transfected with the apo A-I reporter plasmid and the hepatocyte nuclear factors 3α and ß expression plasmids. The apo A-I levels in conditioned media from HepG2 cells, apo A-I mRNA levels, and apo A-I promoter activity increased significantly following treatment with 1.0, 3.0, and 10 mmol/L nicotinic acid. Nicotinic acid-induced promoter activity required a region of the apo A-I gene located between -170 and -186 base pairs. Exogenous overexpression of the hepatocyte nuclear factors 3α and ß had no additive effect on apo A-I promoter. Apolipoprotein A-I concentrations in conditioned media and the apo A-I promoter activity were also significantly increased in Caco-2 intestinal cells. Nicotinic acid may increase apo A-I protein synthesis in the liver and small intestine. Induction of apo A-I gene by nicotinic acid requires a nicotinic acid responsive element in the apo A-I promoter.

24, 25-dihydroxycholecalciferol but not 25-hydroxycholecalciferol suppresses apolipoprotein A-I gene expression.

AIMS: Ligands for the vitamin D receptor (VDR) regulate apolipoprotein A-I (apo A-I) gene expression in a tissue-specific manner. The vitamin D metabolite 24, 25-dihydroxycholecalciferol (24, 25-(OH)(2)D(3)) has been shown to possess unique biological effects. To determine if 24, 25-(OH)(2)D(3) modulates apo A-I gene expression, HepG2 hepatocytes and Caco-2 intestinal cells were treated with 24, 25-(OH)(2)D(3) or its precursor 25-OHD(3). MAIN METHODS: Apo A-I protein levels and mRNA levels were measured by Western and Northern blotting, respectively. Changes in apo A-I promoter activity were measured using the chlorampenicol acetytransferase assay. KEY FINDINGS: Treatment with 24, 25-(OH)(2)D(3), but not 25-OHD(3), inhibited apo A-I secretion in HepG2 and Caco-2 cells and apo A-I mRNA levels and apo A-I promoter activity in HepG2 cells. To determine if 24, 25-(OH)(2)D(3) represses apo A-I gene expression through site A, the nuclear receptor binding element that is essential for VDRs effects on apo A-I gene expression, HepG2 cells were transfected with plasmids containing or lacking site A. While the site A-containing plasmid was suppressed by 24, 25-(OH)(2)D(3), the plasmid lacking site A was not. Likewise, treatment with 24, 25-(OH)(2)D(3) suppressed reporter gene expression in cells transfected with a plasmid containing site A in front of a heterologous promoter. Finally, antisense-mediated VDR depletion failed to reverse the silencing effects of 24, 25-(OH)(2)D(3) on apo A-I expression. SIGNIFICANCE: These results suggest that the vitamin D metabolite 24, 25-(OH)(2)D(3) is an endogenous regulator of apo A-I synthesis through a VDR-independent signaling mechanism.

Hyperglycemia-induced endoplasmic reticulum stress in endothelial cells.

OBJECTIVE: Hyperglycemia-induced endothelial cell dysfunction in vascular disease can occur due to increased oxidative stress and a concomitant increase in endoplasmic reticulum (ER) stress. To investigate whether these cellular stresses are independent or causally linked, we determined whether or not specific glycolytic intermediates that induce oxidative stress also induce ER stress. METHODS: Human umbilical vein endothelial cells were treated with dextrose, partially metabolizable (e.g., fructose and galactose) and non-metabolizable sugars (e.g., 3-O-methyglucose), and various intermediates of the glycolytic and tricarboxylic acid pathways. Activation of the unfolded protein response and subsequent generation of ER stress was measured by the ER stress-responsive alkaline phosphatase method, and superoxide (SO) generation was measured using the hydro-ethidene-fluorescence method. The mitochondrial origin of the SO and the generation of ER stress by dextrose and the intermediate metabolites were confirmed with experiments using allopurinol and diphenyleneiodonium chloride to block SO generation by xanthine oxidase and nicotinamide adenosine dinucleotide phosphate oxidase, respectively. RESULTS: Although ER stress could be induced by glycolytic intermediates up to and including pyruvate, the SO generation occurred in the presence of glycolytic and mitochondrial metabolites. CONCLUSION: Although the mitochondria are the site of signals generated by dextrose to initiate oxidative stress, the dextrose-induced ER stress, unlike SO generation, does not require pyruvate oxidation in the mitochondria.

Effects of antioxidants on glucose-induced oxidative stress and endoplasmic reticulum stress in endothelial cells.

AIM: Hyperglycemia-induced endothelial cell dysfunction can be the result of increased oxidative stress and concomitant increase in endoplasmic reticulum (ER) stress. To test the extent of coupling between these two stresses, the effect of antioxidant vitamins on glucose-induced oxidative stress and ER stress in endothelial cells were studied. METHODS: Human umbilical vein endothelial cells (HUVEC) were treated with physiological (5.5mM) or supra-physiological (27.5mM) dextrose concentrations, and ER stress and oxidative stress were measured. Additional experiments were carried out in HUVEC over-expressing exogenous glucose transporter-1 (Glut-1) and treated with 5.5mM dextrose. RESULTS: Supra-physiological dextrose concentrations increased both ER stress and oxidative stress. However, while oxidative stress could be effectively inhibited with alpha-tocopherol and ascorbic acid, these antioxidants had no effect on ER stress. Increasing intracellular glucose levels by exogenous expression of Glut-1 in endothelial cells also increased oxidative stress and ER stress. Whereas the oxidative stress in these cells was reduced with alpha-tocopherol and ascorbic acid and dimethylsulfoxide, the ER stress could not be ameliorated with alpha-tocopherol and ascorbic acid. CONCLUSIONS: These results indicate that ER stress can be uncoupled from oxidative stress and antioxidants can ameliorate the latter without altering the ER stress induced by hyperglycemia.