Inhibition of apolipoprotein A-I gene expression by obesity-associated endocannabinoids

Obesity is associated with increased serum endocannabinoid (EC) levels and decreased high-density lipoprotein cholesterol (HDLc). Apolipoprotein A-I (apo A-I), the primary protein component of HDL is expressed primarily in the liver and small intestine. To determine whether ECs regulate apo A-I gene expression directly, the effect of the obesity-associated ECs anandamide and 2-arachidonylglycerol on apo A-I gene expression was examined in the hepatocyte cell line HepG2 and the intestinal cell line Caco-2. Apo A-I protein secretion was suppressed nearly 50% by anandamide and 2-arachidonoylglycerol in a dose-dependent manner in both cell lines. Anandamide treatment suppressed both apo A-I mRNA and apo A-I gene promoter activity in both cell lines. Studies using apo A-I promoter deletion constructs indicated that repression of apo A-I promoter activity by anandamide requires a previously identified nuclear receptor binding site designated as site A. Furthermore, anandamide-treatment inhibited protein-DNA complex formation with the site A probe. Exogenous over expression of cannabinoid receptor 1 (CBR1) in HepG2 cells suppressed apo A-I promoter activity, while in Caco-2 cells, exogenous expression of both CBR1 and CBR2 could repress apo A-I promoter activity. The suppressive effect of anandamide on apo A-I promoter activity in Hep G2 cells could be inhibited by CBR1 antagonist AM251 but not by AM630, a selective and potent CBR2 inhibitor. These results indicate that ECs directly suppress apo A-I gene expression in both hepatocytes and intestinal cells, contributing to the decrease in serum HDLc in obese individuals.     

Intestinal apolipoprotein A-IV gene transcription is controlled by two hormone-responsive elements: a role for hepatic nuclear factor-4 isoforms

In the small intestine, the expression of the apolipoprotein (apo) C-III and A-IV genes is restricted to the enterocytes of the villi. We have previously shown that, in transgenic mice, specific expression of the human apo C-III requires a hormone-responsive element (HRE) located in the distal region of the human apoA-IV promoter. This HRE binds the hepatic nuclear factors (HNF)-4alpha and gamma. Here, intraduodenal injections in mice and infections of human enterocytic Caco-2/TC7 cells with an adenovirus expressing a dominant-negative form of HNF-4alpha repress the expression of the apoA-IV gene, demonstrating that HNF-4 controls the apoA-IV gene expression in enterocytes. We show that HNF-4alpha and gamma functionally interact with a second HRE present in the proximal region of the human apoA-IV promoter. New sets of transgenic mice expressing mutated forms of the promoter, combined with the human apo C-III enhancer, demonstrate that, whereas a single HRE is sufficient to reproduce the physiological cephalo-caudal gradient of apoA-IV gene expression, both HREs are required for expression that is restricted to villi. The combination of multiple HREs may specifically recruit regulatory complexes associating HNF-4 and either coactivators in villi or corepressors in crypts.     

Acute suppression of apo B secretion by insulin occurs independently of MTP

Secretion of apolipoprotein (apo) B-containing lipoproteins by the liver depends mainly upon apo B availability and microsomal triglyceride transfer protein (MTP) activity and is subject to insulin regulation. Hepatic MTP mRNA expression is negatively regulated by insulin which correlates with inhibition of apo B secretion suggesting that insulin might suppress apo B secretion through an MTP-dependent mechanism. To investigate this possibility, we examined the acute effect of insulin on hepatic MTP expression and activity levels in vivo utilizing apobec-1^−/− mice. Insulin did not significantly alter hepatic MTP mRNA levels or lipid transfer activity 2 h following injection, but suppressed expression of genes important in gluconeogenesis. To study the specific role of MTP, we expressed human MTP (hMTP) in primary rat hepatocytes using adenoviral gene transfer. Increased expression of hMTP resulted in a 47.6 ± 17.9% increase in total apo B secreted. Incubation of hepatocytes with insulin suppressed apo B secretion by 50.1 ± 10.8% in cells over-expressing hMTP and by 53.0 ± 12.4% in control transfected hepatocytes. Results indicate that even under conditions of increased hepatic apo B secretion mediated by MTP, responsiveness of hepatocytes to insulin to suppress apo B secretion is maintained.     

Lipoprotein secretion by the human hepatoma cell line Hep G2: differential rates of accumulation of apolipoprotein B and lipoprotein lipids in tissue culture media in response to albumin, glucose and oleate

Serum low-density lipoprotein (LDL) concentration is a major determinant of susceptibility to the development of atherosclerosis. A major component of the protein moiety of LDL and its precursor very-low-density lipoprotein is apolipoprotein B (apo B). The human hepatoma cell line, Hep G2, was used as a model for the investigation of mechanisms which control hepatic secretion of the apo B and lipid components of lipoproteins. Using a sensitive immunoradiometric assay for apo B developed in this laboratory, we showed that bovine serum albumin inhibited and glucose, and fatty acids enhanced the rate of accumulation of apo B in the culture medium of Hep G2 cells. However, these substances did not necessarily affect LDL lipids in the same way as apo B. This finding appeared to be due to Hep G2 cells expressing lipase activities which led to triacylglycerol and phospholipid hydrolysis and lipid reuptake. Reuptake of apo B also occurred, but its rate of accumulation in the culture medium suggested it was a closer reflection of its true secretory rate.     

Cell-specific involvement of HNF-1beta in alpha(1)-antitrypsin gene expression in human respiratory epithelial cells

The synergistic action of hepatocyte nuclear factor (HNF)-1alpha and HNF-4 plays an important role in expression of the alpha(1)-antitrypsin (alpha(1)-AT) gene in human hepatic and intestinal epithelial cells. Recent studies have indicated that the alpha(1)-AT gene is also expressed in human pulmonary alveolar epithelial cells, a potentially important local site of the lung antiprotease defense. In this study, we examined the possibility that alpha(1)-AT gene expression in a human pulmonary epithelial cell line H441 was also directed by the synergistic action of HNF-1alpha and HNF-4 and/or by the action of HNF-3, which has been shown to play a dominant role in gene expression in H441 cells. The results show that alpha(1)-AT gene expression in H441 cells is predominantly driven by HNF-1beta, even though HNF-1beta has no effect on alpha(1)-AT gene expression in human hepatic Hep G2 and human intestinal epithelial Caco-2 cell lines. Expression of alpha(1)-AT and HNF-1beta was also demonstrated in primary cultures of human respiratory epithelial cells. HNF-4 has no effect on alpha(1)-AT gene expression in H441 cells, even when it is cotransfected with HNF-1beta or HNF-1alpha. HNF-3 by itself has little effect on alpha(1)-AT gene expression in H441, Hep G2, or Caco-2 cells but tends to have an upregulating effect when cotransfected with HNF-1 in Hep G2 and Caco-2 cells. These results indicate the unique involvement of HNF-1beta in alpha(1)-AT gene expression in a cell line and primary cultures derived from human respiratory epithelium.     

Effects of coumestrol on lipid and glucose metabolism as a farnesoid X receptor ligand

In the course of an effort to identify novel agonists of the farnesoid X receptor (FXR), coumestrol was determined to be one such ligand. Reporter and in vitro coactivator interaction assays revealed that coumestrol bound and activated FXR. Treatment of Hep G2 cells with coumestrol stimulated the expression of FXR target genes, thereby regulating the expression of target genes of the liver X receptor and hepatocyte nuclear factor-4α. Through these actions, coumestrol is expected to exert beneficial effects on lipid and glucose metabolism.     

Effect of 25-hydroxycholesterol and bile acids on the regulation of cholesterol metabolism in Hep G2 cells.

The effect of 25-hydroxycholesterol (25-OH-cholesterol) and chenodeoxycholic (CDC) acid on apoprotein secretion, low-density lipoprotein receptor activity, and [3H]triacylglycerol secretion in Hep G2 cells was studied. Both 25-OH-cholesterol and CDC acid increased the secretion of apolipoprotein (apo) E by Hep G2 cells. The secretion of apo A-I was slightly lowered (less than 10% disease). The maximal increase in apo E secretion was observed in culture medium containing 2 micrograms of 25-OH-cholesterol/ml or 10 micrograms of CDC acid/ml plus 10% fetal calf serum. Cholesterol, 7-OH-cholesterol and other bile acids were ineffective in inducing increases in apo E secretion. Another cholesterol synthesis inhibitor, mevinolin, was also ineffective in generating an increase in apoprotein secretion. The data indicated a specific interaction between 25-OH-cholesterol or CDC acid and apo E secretion in Hep G2 cells. Cholesterol synthesis, as measured by the incorporation of [14C]acetic acid into sterols, was repressed in Hep G2 cells in the presence of 25-OH-cholesterol (17% of control value). CDC acid, on the other hand, increased [14C]acetic acid incorporation (156% of control value). The number of LDL receptors in Hep G2 cells was decreased after incubation with 25-OH-cholesterol (62% of control value), but increased significantly after incubation with CDC acid (149% of control value). The secretion of [3H]triacylglycerol by Hep G2 cells incubated with 25-OH-cholesterol was greatly increased (248% of control value). On the contrary, CDC acid did not cause any increase in [3H]triacylglycerol secretion. The above results suggest that 25-OH-cholesterol and CDC acid have different effects on lipid metabolism in Hep G2 cells. The mRNA levels of apo E increased in cells preincubated with 25-OH-cholesterol and CDC acid, which suggested that the increase in apo E secretion is at least partly due to an increase in synthesis.