Aldose Reductase Regulates Hepatic Peroxisome Proliferator-activated Receptor α Phosphorylation And Activity To Affect Lipid Homeostasis

Aldose reductase (AR) is implicated in the development of a number of diabeticcomplications but the underlying mechanisms remain to be fully elucidated. Weperformed this study to determine whether and how AR might influence hepaticperoxisome proliferator-activated receptorα(PPARα) activity and lipid metabolism.We first constructed an AR over-expressing vector, pFLAG-mAR, andco-transfected it with a reporter plasmid PPRE-tk-Luc containing peroxisomeproliferator response element (PPRE) into mouse hepatocyte AML12 cells in thepresence of a PPARγantagonist G3335. These transfection studies showed that ARover-expression caused strong suppression of PPARα/δactivity (74%, P＜0.001).These suppressive effects were attenuated by selective AR inhibitor (ARI) zopolrestat.The AR-induced reduction in PPARα/δactivities, on the other hand, was associatedwith down-regulated mRNA expression of acyl-CoA oxidase (ACO) (34.3%, P＜0.01)and carnitine palmitoyl transferase-1 (CPT-1) (23.3%, P＜0.05), two PPARαtargetgenes critical for fatty acid oxidation, suggesting AR is involved in the modulation ofhepatic PPARαactivity to affect lipid metabolism. It was further demonstrated byWestern blot that AR over-expression greatly increased phosphorylated PPARαlevels(2.2 folds for phospho-Serine-12 PPARα, P＜0.001 and 2.1 folds forphospho-Serine-21 PPARα, P＜0.05), which is consistent with down-regulatedPPARαactivity. Paralleling this increase, there was a greatly increasedphosphorylation of both ERK1 (14 folds, P＜0.001) and ERK2 (4.1 folds, P＜0.01),suggesting that the ERK-MAPK signaling pathway contributed to the increasedPPARαphosphorylation. In accordance with this, AR-induced suppression of PPARαwas attenuated (78% of the control level, P＜0.001) by treatment with inhibitor forERK1/2 but not that for PI3K, p38, JNK and PKC. Importantly, similar effects wereobserved for cells exposed to 25 mM glucose. AR was up-regulated in AML 12 cellsafter treated with 25 mM glucose, while PPARαtranscriptional activity wassignificantly suppressed, and that was associated with increased phosphorylation ofPPARαand ERK1/2. Knock down of AR by siRNA dephosphorylated PPARαand ERK1/2.We investigated effects of AR on PPARαin streptozotoxin (STZ)-induceddiabetic C57BL/6 mice. Our results showed that ARI treatment or genetic deficiencyof AR resulted in significant dephosphorylation of both hepatic PPARαand ERK1/2.With the dephosphorylation of PPARα, ARI treatment caused a 44% up-regulation ofhepatic ACO mRNA expression (P＜0.01) and a 34.8% down-regulation ofapolipoprotein ApoC3 mRNA expression (P＜0.01) in wild type STZ-diabetic mice,and that was associated with substantial reductions in blood triglyceride (TG) andnon-esterified fatty acid (NEFA) levels. We further investigated effects of AR onPPARαin Type 2 diabetes mellitus models. In db/db mice, ARI treatment alsoresulted in significant dephosphorylation of both PPARαand ERK1/2. And hepaticACO and apolipoprotein ApoA5 mRNA expression increased 92% (P＜0.01) and73% (P＜0.01) respectively. Liver TG levels decreased significantly and serum TGlevels also decreased significantly after ARI treatment. Oil red O staining of liversection also showed that neutral lipid contents were reduced after ARI treatment indb/db mice.Together our data indicate that AR plays an important role in the regulation ofhepatic PPARαphosphorylation and activity and lipid homeostasis. A significantportion of the AR-induced modulation is achieved through ERK1/2 signaling.