LDL and UV-oxidized LDL induce upregulation of iNOS and NO in unstimulated J774 macrophages and HUVEC.

Oxidized low-density lipoprotein (LDL) diminishes NO production from activated macrophages. The interaction between LDL and inactivated macrophages is neglected and controversial. This study examines the effect of LDL, 7-oxysterols and iron compounds on NO production in unstimulated J774 macrophages. J774 cells and human umbilical vein endothelial cells (HUVEC) were either incubated for 24 h with native LDL (LDL) or ultraviolet (UV)-oxidized LDL (UVoxLDL), in the absence or presence of an inducible nitric oxide synthase (iNOS)- or an endothelial constitutive nitric oxide synthase (eNOS)-inhibitor. J774 cells were also incubated with lipopolysaccharide (LPS), in the absence or presence of an iNOS- or an eNOS-inhibitor. Nitrite was analysed as a marker of NO production. The mRNA levels of iNOS were evaluated by reverse transcriptase polymerase chain reaction. LDL and UVoxLDL significantly increased NO production from unstimulated J774 macrophages. This increase in NO was accompanied by enhanced expression of iNOS mRNA, and was inhibited by the iNOS inhibitor. Furthermore, NO production was elevated and angiotensin-converting enzyme (ACE) activity was reduced in HUVEC following the exposure to LDL and UVoxLDL. In conclusion, LDL may serve as an important inflammatory activator of macrophages and HUVEC, inducing inducible nitric oxide production but diminishing ACE. After its oxidation, this function of LDL may be further enhanced and may contribute to the regulation and progression of atheroma formation.

Human, but not rat, IRS1 targets to the plasma membrane in both human and rat adipocytes.

Adipocytes are primary targets for insulin control of metabolism. The activated insulin receptor phosphorylates insulin receptor substrate-1 (IRS1), which acts as a docking protein for downstream signal mediators. In the absence of insulin stimulation, IRS1 in rat adipocytes is intracellular but in human adipocytes IRS1 is constitutively targeted to the plasma membrane. Stimulation of adipocytes with insulin increased the amount of IRS1 at the plasma membrane 2-fold in human adipocytes, but >10-fold in rat adipocytes, with the same final amount of IRS1 at the plasma membrane in cells from both species. Cross-transfection of rat adipocytes with human IRS1, or human adipocytes with rat IRS1, demonstrated that the species difference was due to the IRS1 protein and not the cellular milieus or posttranslational modifications. Chimeric IRS1, consisting of the conserved N-terminus of rat IRS1 with the variable C-terminal of human IRS1, did not target the plasma membrane, indicating that subtle sequence differences direct human IRS1 to the plasma membrane.

Angiotensin type 1 receptor antagonists induce human in-vitro adipogenesis through peroxisome proliferator-activated receptor-gamma activation.

OBJECTIVE: In clonal animal cells, certain angiotensin receptor blockers (ARB) activate the peroxisome proliferator-activated receptor-gamma (PPARgamma). The aim of this work was to validate that observation in human cells and humans. METHODS: We investigated the induction of in-vitro adipogenesis and the activation of PPARgamma-target genes, adiponectin and lipoprotein lipase, by ARB in human preadipocytes. We also studied PPARgamma response-element-driven luciferase reporter gene activation in human adipocytes. Finally, we treated 14 obese men for 10 days with placebo crossed over with 150 mg/day irbesartan. Subcutaneous fat was analyzed for mRNA expression of adiponectin and lipoprotein lipase. RESULTS: Telmisartan and irbesartan, and to a lesser degree losartan, induced adipogenesis and activated PPARgamma-target genes. This stimulation of PPARgamma-target genes was prevented by the PPARgamma antagonist GW9662. Eprosartan had no effect. Paradoxically, all ARB activated the luciferase reporter gene. PPARgamma activity increased approximately two-fold with pioglitazone and 1.5-fold with the ARB in all assays. In the cross-over clinical study, irbesartan lowered blood pressure but had no effect on adiponectin or lipoprotein lipase mRNA expression. CONCLUSIONS: Our data are the first to show that ARB induce adipogenesis and PPARgamma-target gene expression in human adipocytes. Pharmacokinetic differences may contribute to the heterogeneous effects on metabolism and preadipocyte differentiation. In humans, larger doses of ARB, longer treatments, or both may be required to activate PPARgamma in adipose cells.

PPAR-gamma response element activity in intact primary human adipocytes: effects of fatty acids.

OBJECTIVE: We studied the activity and regulation of the peroxisome proliferator-activated receptor-gamma response element (PPRE) in primary human adipocytes. METHODS: We transfected primary human adipocytes with a plasmid-encoding firefly luciferase cDNA under control of a PPRE from the acyl-coenzyme A oxidase gene by using our newly developed electroporation-based method. Several fatty acids were added to the fat cells to study potential activation of peroxisome proliferator-activated receptor-gamma. RESULTS: Cells responded maximally to 5 microM of rosiglitazone at a 5.1 +/- 1.4-fold over basal increase in luciferase activity. There was a positive correlation between body mass index and the response to 5 microM of rosiglitazone (r = 0.36, P = 0.03). Patients with type 2 diabetes had similar basal PPRE activity but responded more strongly to 5 microM of rosiglitazone than did non-diabetic subjects (10.2 +/- 5-fold and 5.4 +/- 1-fold over basal increase, respectively, P < 0.0001). Among saturated fatty acids, lauric acid was without effect, but 10 microM of palmitic or stearic acid increased PPRE activity 20% to 35% above basal levels. Monounsaturated palmitoleic acid at 1 microM induced a PPRE transcriptional activity that corresponded to half the therapeutic levels of rosiglitazone. CONCLUSION: Adipocytes from obese subjects and patients with type 2 diabetes responded particularly strongly to the effect of rosiglitazone on PPRE. Because fatty acids in the diet can affect the transcriptional activity of peroxisome proliferator-activated receptor-gamma over decades, the stimulation induced by stearic and palmitoleic acids can affect insulin sensitivity and, hence, cardiovascular morbidity and mortality in humans.