We previously proposed that the production of hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) is a key event in the development of diabetes complications. The association between the pathogenesis of diabetes and its complications and mitochondrial biogenesis has been recently reported. Because metformin has been reported to exert a possible additional benefit in preventing diabetes complications, we investigated the effect of metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on mtROS production and mitochondrial biogenesis in cultured human umbilical vein endothelial cells. Treatment with metformin and AICAR inhibited hyperglycemia-induced intracellular and mtROS production, stimulated AMP-activated protein kinase (AMPK) activity, and increased the expression of peroxisome proliferator-activated response-␥ coactivator-1␣ (PGC-1␣) and manganese superoxide dismutase (MnSOD) mRNAs. The dominant negative form of AMPK␣1 diminished the effects of metformin and AICAR on these events, and an overexpression of PGC-1␣ completely blocked the hyperglycemiainduced mtROS production. In addition, metformin and AICAR increased the mRNA expression of nuclear respiratory factor-1 and mitochondrial DNA transcription factor A (mtTFA) and stimulated the mitochondrial proliferation. Dominant negative-AMPK also reduced the effects of metformin and AICAR on these observations. These results suggest that metformin normalizes hyperglycemia-induced mtROS production by induction of MnSOD and promotion of mitochondrial biogenesis through the activation of AMPK-PGC-1␣ pathway. Diabetes 55:120 -127, 2006

Hyperglycemia increases the production of reactive oxygen species (ROS) from the mitochondrial electron transport chain in bovine endothelial cells. Because several studies have postulated a role for prostaglandins (PGs) in the glomerular hyperfiltration seen in early diabetes, we evaluated the effect of mitochondrial ROS on expression of the inducible isoform of cyclooxygenase (COX-2) in cultured human mesangial cells (HMCs). We first confirmed that incubation of HMC with 30 mmol/l glucose significantly increased COX-2 mRNA but not COX-1 mRNA, compared with 5.6 mmol/l glucose. Similarly, incubation of HMCs with 30 mmol/l glucose significantly increased mitochondrial membrane potential, intracellular ROS production, COX-2 protein expression, and PGE2 synthesis, and these events were completely suppressed by thenoyltrifluoroacetone or carbonyl cyanide m-chlorophenylhydrazone, inhibitors of mitochondrial metabolism, or by overexpression of uncoupling protein-1 or manganese superoxide dismutase. Furthermore, increased expression of COX-2 mRNA and protein was confirmed in glomeruli of streptozotocin-induced diabetic mice. In addition, hyperglycemia induced activation of the COX-2 gene promoter, which was completely abrogated by mutation of two nuclear factor κB (NF-κB) binding sites in the promoter region. Our results suggest that hyperglycemia increases mitochondrial ROS production, resulting in NF-κB activation, COX-2 mRNA induction, COX-2 protein production, and PGE2 synthesis. This chain of events might contribute to the pathogenesis of diabetic nephropathy.

Abstract-Both statins and peroxisome proliferator-activated receptor (PPAR)␥ ligands have been reported to protect against the progression of atherosclerosis. In the present study, we investigated the effects of statins on PPAR␥ activation in macrophages. Statins increased PPAR␥ activity, which was inhibited by mevalonate, farnesylpyrophosphate, or geranylgeranylpyrophosphate. Furthermore, a farnesyl transferase inhibitor and a geranylgeranyl transferase inhibitor mimicked the effects of statins. Statins inhibited the membrane translocations of Ras, RhoA, Rac, and Cdc42, and overexpression of dominant-negative mutants of RhoA (DN-RhoA) and Cdc42 (DN-Cdc42), but not of Ras or Rac, increased PPAR␥ activity. Statins induced extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) activation. However, DN-RhoA and DN-Cdc42 activated p38 MAPK, but not ERK1/2. ERK1/2-or p38 MAPK-specific inhibitors abrogated statin-induced PPAR␥ activation. Statins induced cyclooxygenase (COX)-2 expression and increased intracellular 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) levels through ERK1/2-and p38 MAPK-dependent pathways, and inhibitors or small interfering RNA of COX-2 inhibited statin-induced PPAR␥ activation. Statins also activate PPAR␣ via COX-2-dependent increases in 15d-PGJ 2 levels. We further demonstrated that statins inhibited lipopolysaccharide-induced tumor necrosis factor ␣ or monocyte chemoattractant protein-1 mRNA expression, and these effects by statins were abrogated by the PPAR␥ antagonist T0070907 or by small interfering RNA of PPAR␥ or PPAR␣. Statins also induced ATP-binding cassette protein A1 or CD36 mRNA expression, and these effects were suppressed by small interfering RNAs of PPAR␥ or PPAR␣. In conclusion, statins induce COX-2-dependent increase in 15d-PGJ 2 level through a RhoA-and Cdc42-dependent p38 MAPK pathway and a RhoA-and Cdc42-independent ERK1/2 pathway, thereby activating PPAR␥. Statins also activate PPAR␣ via COX-2-dependent pathway. These effects of statins may explain their antiatherogenic actions. (Circ Res. 2007;100:1442-1451.) Key Words: cyclooxygenase Ⅲ MAPK Ⅲ macrophages Ⅲ PPAR Ⅲ statins 3 -Hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors (statins) are known to reduce the incidence of cardiovascular events and death, and these benefits are mainly caused by their lipid-lowering effects. 1 However, recent evidence has suggested that the beneficial effects by statins are independent of their cholesterol-lowering effects. 2 Cholesterol is synthesized via the isoprenoid biosynthetic pathway. 3 In this pathway, isopentenyl-PP is the basic isoprene unit used for synthesis of all subsequent isoprenoids. 3 Among the isoprenoids, farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) serve as important lipid attachments for several proteins, including the small GTP-binding protein Ras and Ras-like proteins, such as Rho, Rac, and Cdc42, whose proper membrane localization and function are dependent on isoprenylation. 3 The pleio...

OBJECTIVE -To evaluate urinary 8-hydroxydeoxyguanosine (8-OHdG) as a marker for the progression of diabetic macroangiopathic complications. RESEARCH DESIGN AND METHODS -The content of urinary 8-OHdG, common carotid intima-media thickness (IMT), the coronary heart disease (CHD) risk score, the severity of diabetic retinopathy, and urinary albumin excretion were examined in 96 patients with type 2 diabetes, including 32 patients who had been nominated for the Kumamoto Study [Shichiri M, et al. Diabetes Care 23 (Suppl 2):B21-B29, 2000]. In addition, the patients from the Kumamoto Study were further evaluated regarding the effect of intensive insulin therapy on urinary 8-OHdG excretion.RESULTS -The urinary 8-OHdG:creatinine ratio (U8-OHdG) was 2.5-fold higher in patients with increased HbA 1c than in those with normal HbA 1c (P Ͻ 0.05). In addition, U8-OHdG was 2.3-fold higher in patients with increased IMT (P Ͻ 0.005). A similar result was observed between U8-OHdG and CHD risk score (P Ͻ 0.01). U8-OHdG was significantly higher in patients with simple retinopathy (P Ͻ 0.05) and those with advanced retinopathy (P Ͻ 0.01) than in patients without retinopathy. Similarly, U8-OHdG was significantly higher in patients with albuminuria (P Ͻ 0.01). Furthermore, in the Kumamoto Study, U8-OHdG was significantly lower in the multiple insulin injection therapy group compared with the conventional insulin injection therapy group (P Ͻ 0.01).CONCLUSIONS -Hyperglycemia independently increases 8-OHdG in patients with type 2 diabetes. 8-OHdG is a useful biomarker of not only microvascular but also macrovascular complications in patients with type 2 diabetes. Diabetes Care 26:1507-1512, 2003V ascular complications are the leading cause of morbidity and mortality in patients with diabetes. In adult patients with diabetes, the risk of cardiovascular disease is three-to fivefold greater than in nondiabetic subjects despite controlling for other known risk factors for cardiovascular disease (1). In addition, diabetic microangiopathy still represents one of the main causes of blindness (2), terminal renal failure (3), and amputation (4).The outcomes of the Diabetes Control and Complication Trial (5), the Kumamoto Study (6 -8), and the U.K. Prospective Diabetes Study (9) seem to have effectively resolved the long debate over whether chronic hyperglycemia is an important cause of diabetic vascular complications. Furthermore, the Diabetes Insulin-Glucose in Acute Myocardial Infarction Study showed that intensive insulin treatment was associated with a lower mortality rate than conventional insulin treatment in subjects with acute myocardial infarction (10). It was also reported that early atherosclerosis could be retarded by improved glycemic control in patients with type 1 diabetes (11). Therefore, hyperglycemia represents a major contributing factor to not only microvascular complications in diabetes but also macrovascular complications. Next to this, the longest-running controversy yet to be resolved concerns the identification of the me...

Tumor necrosis factor (TNF)-␣ inhibits insulin action;however, the precise mechanisms are unknown. It was reported that TNF-␣ could increase mitochondrial reactive oxygen species (ROS) production, and apoptosis signalregulating kinase 1 (ASK1) was reported to be required for TNF-␣-induced apoptosis. Here, we examined roles of mitochondrial ROS and ASK1 in TNF-␣-induced impaired insulin signaling in cultured human hepatoma (Huh7) cells. Using reduced MitoTracker Red probe, we confirmed that TNF-␣ increased mitochondrial ROS production, which was suppressed by overexpression of either uncoupling protein-1 (UCP)-1 or manganese superoxide dismutase (Mn-SOD). TNF-␣ significantly activated ASK1, increased serine phosphorylation of insulin receptor substrate (IRS)-1, and decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, and all of these effects were inhibited by overexpression of either UCP-1 or MnSOD. Similar to TNF-␣, overexpression of wild-type ASK1 increased serine phosphorylation of IRS-1 and decreased insulin-stimulated tyrosine phosphorylation of IRS-1, whereas overexpression of dominant-negative ASK1 ameliorated these TNF-␣-induced events. In addition, TNF-␣ activated c-jun NH 2 -terminal kinases (JNKs), and this observation was partially inhibited by overexpression of UCP-1, MnSOD, or dominant-negative ASK1. These results suggest that TNF-␣ increases mitochondrial ROS and activates ASK1 in Huh7 cells and that these TNF-␣-induced phenomena contribute, at least in part, to impaired insulin signaling.