Insulin-mediated suppression of apolipoprotein B mRNA translation requires the 5' UTR and is characterized by decreased binding of an insulin-sensitive 110-kDa 5' UTR RNA-binding protein.

Insulin has been shown to acutely regulate hepatic apolipoprotein B (apoB) secretion at both translational and post-translational levels; however, mechanisms of apoB mRNA translational control are largely unknown. Recent studies of apoB untranslated regions (UTRs) revealed a potentially important role for cis-trans interactions at the 5' and 3' UTRs. In the present paper, deletion constructs of the UTR regions of apoB revealed that the 5' UTR was necessary and sufficient for insulin to inhibit synthesis of apoB15. Metabolic radiolabeling and in vitro translation experiments in the presence of protease inhibitors confirmed that the effect of insulin on the apoB 5' UTR was translational in nature. Using the nondenaturing electrophoretic mobility shift assay (EMSA), protein-RNA complexes were detected binding to the apoB 5' and 3' UTRs. Denaturing EMSA identified a 110-kDa protein interacting at the 5' UTR. Nondenaturing EMSA determined that insulin altered binding of large protein complexes to the 5' UTR. Binding specificity was determined by competition with both specific and nonspecific competitors. Insulin treatment decreased binding of the 110-kDa protein to the 5' UTR as visualized by EMSA. Absence of insulin increased binding of this trans-acting factor to the 5' UTR by 2-fold. Analysis of the 3' UTR showed no significant insulin-mediated changes in binding of trans-acting factors. We thus propose the existence of a novel RNA-binding insulin-sensitive factor that binds to the 5' UTR and may regulate apoB mRNA translation. Perturbations in hepatic insulin signaling as observed in insulin-resistant states may alter cis-trans interactions at the 5' UTR, leading to alterations in the rate of apoB mRNA translation, thus contributing to apoB-lipoprotein overproduction.

Translational control of apolipoprotein B mRNA: regulation via cis elements in the 5' and 3' untranslated regions.

Translational control of apolipoprotein B (apoB) mRNA has been previously documented; however, the molecular mechanisms that govern translation of apoB mRNA are unknown. We investigated the role of the untranslated regions (UTR) in the regulation of apoB mRNA translation first by analyzing apoB UTR sequences using M-fold, a program used to predict RNA secondary structure. M-fold analysis revealed hairpin-like elements within the 5'UTR and 3'UTR of apoB mRNA with potential to form stable secondary structure. Luciferase (LUC) reporter assays were conducted to assess the biological activity of the putative RNA motifs within the UTR sequences by transiently transfecting HepG2 cells with chimeric mRNAs containing the 5' and/or 3' apoB UTRs linked to a LUC reporter gene. We observed statistically significant increases in LUC activity for the 5'UTRpGL3 and 5'/3'UTRpGL3 constructs. LUC mRNA levels remained constant for all constructs, suggesting that increased LUC activity was likely posttranscriptional in nature. When RNA isolated from transfected cells was translated in vitro, parallel increases in translatable LUC activity were observed. We also examined the role of UTR sequences within the context of the apoB coding sequence, using constructs containing the N-terminal 15% of apoB (apoB15). We observed a 40% and 25% increase in total protein mass with the 5'UTR-apoB15 construct and the 5'UTR-apoB15-3'UTR, respectively, over the control construct with no apoB UTR, with only a slight stimulation observed for apoB15 3'UTR. Radiolabeling analysis of apoB15 synthetic rate showed a more striking 4.5-fold stimulation of protein synthesis by 5'UTR while addition of both UTRs caused a 3.1-fold stimulation over the control construct. Deletion mutant analysis revealed that the stimulatory effect of the 5'UTR on apoB mRNA translation may be dependent on specific hairpin elements formed within the 5'UTR secondary structure. Overall, our data suggest that putative 5'UTR motifs are important for optimal translation of the apoB message whereas the presence of the 3'UTR appears to attenuate wild-type expression. Potential cis-trans interactions of these motifs with putative RNA binding proteins/translational factors are likely to govern apoB mRNA translation and protein synthesis and may play an important role in dysregulation of atherogenic lipoprotein production in dyslipidemic states.

Atorvastatin treatment beneficially alters the lipoprotein profile and increases low-density lipoprotein particle diameter in patients with combined dyslipidemia and impaired fasting glucose/type 2 diabetes.

Diabetic dyslipidemia is featured by hypertriglyceridemia, low high-density lipoprotein (HDL) cholesterol levels, and elevated low-density lipoprotein (LDL) cholesterol commonly in the form of small, dense LDL particles. First-line treatment, fibrates versus statins or both, of dyslipidemia in diabetic patients has been the focus of debate. We investigated the potential hypolipidemic effects of atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor with good triglyceride lowering properties, in patients with combined dyslipidemia and evidence of impaired fasting glucose or type 2 diabetes. Twenty patients were recruited for the study, and after a 60-day wash out period, baseline measurements of lipoprotein parameters, LDL particle diameter, and apolipoprotein B (apoB) degradation fragments were obtained. The group was then randomized, in a double-blinded manner, into 2 subgroups. Group A received atorvastatin (80 mg) and group B received placebo daily for 60 days. After the first treatment period, all patients were reanalyzed for the above parameters. The treatment regime then crossed over for the second treatment period in which group A received placebo and group B received atorvastatin (80 mg) daily for 60 days. All parameters were remeasured at the end of the study. Treatment with atorvastatin resulted in a statistically significant reduction in total cholesterol (41%), LDL cholesterol (55%), triglycerides (TG) (32%), and apoB (40%). Mean LDL particle diameter significantly increased from 25.29 +/- 0.24 nm (small, dense LDL subclass) to 26.51 < 0.18 nm (intermediate LDL subclass) after treatment with atorvastatin (n = 20, P <.005). At baseline, LDL particles were predominantly found in the small, dense subclass; atorvastatin treatment resulted in a shift in the profile to the larger and more buoyant LDL subclass. Atorvastatin treatment did not produce consistent changes in the appearance of apoB degradation fragments in plasma. Our results suggest that atorvastatin beneficially alters the atherogenic lipid profile in these patients and significantly decreases the density of LDL particles produced resulting in a shift from small, dense LDL to more buoyant and less atherogenic particles.

Treatment with atorvastatin ameliorates hepatic very-low-density lipoprotein overproduction in an animal model of insulin resistance, the fructose-fed Syrian golden hamster: evidence that reduced hypertriglyceridemia is accompanied by improved hepatic insulin sensitivity.

A novel animal model of insulin resistance, the fructose-fed Syrian golden hamster has been previously documented to exhibit considerable hepatic very-low-density lipoprotein (VLDL) overproduction concomitant with the development of whole body insulin resistance. Here, we investigated whether hepatic lipoprotein overproduction can be ameliorated by treatment with a hydroxymethyl glutaryl conenzyme A (HMG-CoA) reductase inhibitor, atorvastatin, using a series of ex vivo experiments. Hamsters were fed a fructose-enriched diet for 14 days to induce a state of insulin resistance, and then continued on a fructose-enriched diet supplemented with or without 40 mg/kg atorvastatin per day for 14 days. Fructose feeding in the first 2 weeks caused a significant increase in plasma total cholesterol and triglyceride levels. There was a significant decline in plasma triglyceride levels following supplementation with the inhibitor (50% to 59%; P <.05). Experiments with primary hepatocytes revealed a decreased VLDL-apolipoprotein B (apoB) production (37.4% +/- 10.4%; P <.05) in hamsters treated with atorvastatin. Interestingly, atorvastatin treatment partially attenuated (by 23%) the elevated hepatic level of microsomal triglyceride transfer protein (MTP) induced by fructose feeding. There was molecular evidence of improved hepatic insulin sensitivity with atorvastatin treatment based on assessment of the phosphorylation status of the insulin receptor and the expression of protein tyrosine phosphatase-1B. The improvement in insulin signaling was not mediated by a change in hepatic triglyceride accumulation as no significant difference was observed in liver triglyceride levels. Taken together, these data suggest that statins can ameliorate the VLDL-apoB overproduction state observed in a fructose-fed, insulin-resistant hamster model, and may potentially contribute to an enhanced hepatic insulin sensitivity.