S. Yamagishi

Metformin Inhibits Advanced Glycation End Products (AGEs)-induced Renal Tubular Cell Injury by Suppressing Reactive Oxygen Species Generation via Reducing Receptor for AGEs (RAGE) Expression

Advanced glycation end products (AGEs) and their receptor (RAGE) play a role in tubulointerstitial damage in diabetic nephropathy. Recently, metformin has been shown to ameliorate tubular injury both in cell culture and diabetic animal model. However, effects of metformin on AGEs-induced tubular cell apoptosis and damage remain unknown. We examined here whether and how metformin could block the AGEs-RAGE-elicited tubular cell injury in vitro. Gene expression level was evaluated by real-time reverse-transcription polymerase chain reactions. Reactive oxygen species (ROS) generation was measured with dihydroethidium staining. Apoptosis was evaluated by DNA fragmentation and annexin V expression level. AGEs upregulated RAGE mRNA levels and subsequently increased ROS generation and intercellular adhesion molecule-1, monocyte chemoattractant protein-1 and transforming growth factor-β gene expression in human renal proximal tubular cells, all of which were significantly blocked by the treatment of 0.01 and 0.1 mM metformin. Compound C, an inhibitor of AMP-activated protein kinase significantly blocked the effects of metformin on RAGE gene expression and ROS generation in AGEs-exposed tubular cells. Furthermore, metformin dose-dependently inhibited the AGEs-induced apoptotic cell death of tubular cells; 1 mM metformin completely suppressed the pro-apoptotic effects of AGEs in 2 different assay systems. Our present study suggests that metformin could inhibit the AGEs-induced apoptosis and inflammatory and fibrotic reactions in tubular cells probably by reducing ROS generation via suppression of RAGE expression through AMP-activated protein kinase activation. Metformin may protect against tubular cell injury in diabetic nephropathy by blocking the AGEs-RAGE-ROS axis.

Sitagliptin augments protective effects of GLP-1 against advanced glycation end product receptor axis in endothelial cells.

Sitagliptin is a stable inhibitor of dipeptidyl peptidase-IV, a responsible enzyme that mainly inactivates glucagon-like peptide-1 (GLP-1), and now one of the widely used agents for the treatment of diabetes. However, effects of sitagliptin on vascular injury are largely unknown. Since advanced glycation end products (AGEs) and their receptor (RAGE) axis contribute to vascular damage in diabetes, we investigated here whether sitagliptin inhibits the AGE-RAGE-induced endothelial cell damage in vitro. Although effects of 10 pM GLP-1 or 0.5 μM sitagliptin monotherapy on RAGE gene and protein expression were modest, combination therapy completely blocked the AGE-induced increase in RAGE mRNA and protein levels in human umbilical vein endothelial cells (HUVEC). AGEs induced reactive oxygen species (ROS) generation and reduced endothelial nitric oxide synthase (eNOS) mRNA level in HUVEC, both of which were also completely blocked by the treatment with 10 pM GLP-1 and 0.5 μM sitagliptin, but not with GLP-1 or sitagliptin monotherapy. Further, anti-RAGE antibody restored the decrease in eNOS mRNA level in AGE-exposed HUVEC. The present study suggests that sitagliptin augments the effects of GLP-1 on eNOS mRNA level in AGE-exposed HUVEC by suppressing RAGE expression and subsequent ROS generation. Sitagliptin may work as a vasoprotecitve agent in diabetes by blocking the AGE-RAGE axis.

Empagliflozin, an Inhibitor of Sodium-Glucose Cotransporter 2 Exerts Anti-Inflammatory and Antifibrotic Effects on Experimental Diabetic Nephropathy Partly by Suppressing AGEs-Receptor Axis

Advanced glycation end products (AGEs) and receptor RAGE play a role in diabetic nephropathy. We have previously shown that increased glucose uptake into proximal tubular cells via sodium-glucose cotransporter 2 (SGLT2) stimulates oxidative stress generation and RAGE expression, thereby exacerbating the AGE-induced apoptosis in this cell type. However, the protective role of SGLT2 inhibition against the AGE-RAGE-induced renal damage in diabetic animals remains unclear. In this study, we investigated the effects of empagliflozin, SGLT2 inhibitor on AGE-RAGE axis, inflammatory and fibrotic reactions, and tubular injury in the kidney of streptozotocin-induced diabetic rats.Administration of empagliflozin for 4 weeks significantly improved hyperglycemia and HbA1c, and decreased expression levels of AGEs, RAGE, 8-hydroxydeoxyguanosine (8-OHdG), and F4/80, markers of oxidative stress and macrophages, respectively, in the diabetic kidney. Although empagliflozin did not reduce albuminuria, it significantly decreased urinary excretion levels of 8-OHdG and L-fatty acid binding protein, a marker of tubular injury. Moreover, inflammatory and fibrotic gene expression such as monocyte chemoattractant protein-1, intercellular adhesion molecule-1, plasminogen activator inhibitor-1, transforming growth factor-β, and connective tissue growth factor was enhanced in the diabetic kidney, all of which were prevented by empagliflozin. The present study suggests that empagliflozin could inhibit oxidative, inflammatory and fibrotic reactions in the kidney of diabetic rats partly via suppression of the AGE-RAGE axis. Blockade of the increased glucose uptake into renal proximal tubular cells by empagliflozin might be a novel therapeutic target for tubulointerstitial damage in diabetic nephropathy.

Glucose-dependent insulinotropic polypeptide (GIP) inhibits signaling pathways of advanced glycation end products (AGEs) in endothelial cells via its antioxidative properties.

Glucose-dependent insulinotropic polypeptide (GIP) is one of the incretins, a gut hormone secreted from K cells in the intestine in response to food intake. It could be a potential therapeutic target for the treatment of patients with type 2 diabetes. However, effects of GIP on vascular injury remain unknown. Since interaction of advanced glycation end products (AGEs) with their receptor RAGE has been shown to play a crucial role in vascular damage in diabetes, this study investigated whether and how GIP blocked the deleterious effects of AGEs on human umbilical vein endothelial cells (HUVECs). GIP receptor was expressed in HUVECs. GIP, an analogue of cyclic AMP or inhibitors of NADPH oxidase inhibited the AGE-induced reactive oxygen species (ROS) generation in HUVECs. Furthermore, GIP reduced both RAGE mRNA and protein levels in HUVECs. GLP-1 also blocked the AGE-induced increase in mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and plasminogen activator inhibitor-1 in HUVECs. In addition, an antioxidant N-acetylcysteine mimicked the effects of GIP on RAGE and VCAM-1 gene expression in HUVECs. Our present study suggests that GIP could block the signal pathways of AGEs in HUVECs by reducing ROS generation and subsequent RAGE expression probably via GIP receptor-cyclic AMP axis.

Rosuvastatin blocks advanced glycation end products-elicited reduction of macrophage cholesterol efflux by suppressing NADPH oxidase activity via inhibition of geranylgeranylation of Rac-1.

Adenosine triphosphate-binding membrane cassette transporter A1 (ABCA1) and ABCG1 play a crucial role in macrophage cholesterol efflux, which is a novel therapeutic target for atherosclerosis. Advanced glycation end products (AGE) and their receptor RAGE axis is involved in accelerated atherosclerosis in diabetes as well. However, the role of AGE-RAGE axis in macrophage cholesterol efflux is not fully understood. We examined here whether AGE-RAGE axis could impair cholesterol efflux from human macrophage cells, THP-1 cells by suppressing ABCA1 and ABCG1 expression. We further investigated the effects of rosuvastatin on cholesterol efflux from AGE-exposed THP-1 cells. AGE increased reactive oxygen species generation in THP-1 cells, which was completely inhibited by rosuvastatin, anti-RAGE-antibody or diphenylene iodonium chloride (DPI), an inhibitor of NADPH oxidase. The antioxidative effect of rosuvastatin on AGE-exposed THP-1 cells was significantly prevented by geranylgeranyl pyrophosphate (GGPP). AGE decreased ABCA1 and ABCG1 mRNA levels, and subsequently reduced cholesterol efflux from THP-1 cells, which was prevented by GGPP. DPI mimicked the effects of rosuvastain. The results demonstrated that rosuvastatin could inhibit the AGE-induced reduction of THP-1 macrophage cholesterol efflux by suppressing NADPH oxidase activity via inhibition of geranylgeranylation of Rac-1. Our present study provides a novel beneficial aspect of rosuvastatin in diabetes; rosuvastain may prevent the development and progression of atherosclerosis in diabetes by not only reducing serum cholesterol level, but also by improving cholesterol efflux from foam cells of the arterial wall via blocking the harmful effects of AGE on macrophages.

Telmisartan, an Angiotensin II Type 1 Receptor Blocker, Inhibits Advanced Glycation End-product (AGE)-elicited Hepatic Insulin Resistance via Peroxisome Proliferator-activated Receptor-γ Activation

This study examined whether telmisartan, a unique angiotensin II type 1 receptor blocker (ARB) with peroxisome proliferator-activated receptor-γ (PPAR-γ)-modulating activity, improved insulin resistance in advanced glycation end-product (AGE)-exposed human hepatoma (Hep3B) cells. AGE increased phosphorylation of insulin receptor substrate-1 (IRS-1) at serine-307 residues in Hep3B cells. It also decreased tyrosine phosphorylation of IRS-1 and, subsequently, reduced the association of the p85 subunit of phosphatidylinositol 3-kinase with IRS-1 and glycogen synthesis in insulin-exposed Hep3B cells, all of which were inhibited by telmisartan. The insulin-sensitizing properties of telmisartan in AGE-exposed Hep3B cells were significantly blocked by GW9662, an inhibitor of PPAR-γ. Candesartan, another ARB, did not affect AGEs-induced serine phosphorylation of IRS-1 at serine-307 residues in Hep3B cells. Our study suggests that telmisartan could improve AGE-elicited insulin resistance in Hep3B cells by inhibiting serine phosphorylation of IRS-1, at least in part, via activation of PPAR-γ. Telmisartan may play a protective role against hepatic insulin resistance in diabetes.

Tofogliflozin, A Highly Selective Inhibitor of SGLT2 Blocks Proinflammatory and Proapoptotic Effects of Glucose Overload on Proximal Tubular Cells Partly by Suppressing Oxidative Stress Generation

Ninety percent of glucose filtered by the glomerulus is reabsorbed by a sodium-glucose cotransporter 2 (SGLT2), which is mainly expressed on S1 and S2 segment of renal proximal tubules. Since SGLT-2-mediated glucose reabsorption is increased under diabetic conditions, selective inhibition of SGLT2 is a potential therapeutic target for the treatment of diabetes. We have recently shown that an inhibitor of SGLT2 has anti-inflammatory and antifibrotic effects on experimental diabetic nephropathy partly by suppressing advanced glycation end products formation and oxidative stress generation in the kidney. However, the direct effects of SGLT2 inhibitor on tubular cell damage remain unclear. In this study, we investigated the effects of tofogliflozin, a highly selective inhibitor of SGLT2 on oxidative stress generation, inflammatory and proapoptotic reactions in cultured human proximal tubular cells exposed to high glucose. Tofogliflozin dose-dependently suppressed glucose entry into tubular cells. High glucose exposure (30 mM) for 4 and 24 h significantly increased oxidative stress generation in tubular cells, which were suppressed by the treatment of tofogliflozin or an antioxidant N-acetylcysteine (NAC). Monocyte chemoattractant protein-1 (MCP-1) gene expression and apoptotic cell death were induced by 4 h- and 8 day-exposure to high glucose, respectively, both of which were also blocked by tofogliflozin or NAC. The present study suggests that SGLT2-mediated glucose entry into tubular cells could stimulate oxidative stress and evoke inflammatory and proapoptotic reactions in this cell type. Blockade of glucose reabsorption in tubular cells by SGLT2 inhibitor might exert beneficial effects on tubulointerstitial damage in diabetic nephropathy.

Combination Therapy with Nateglinide and Vildagliptin Improves Postprandial Metabolic Derangements in Zucker Fatty Rats

Postprandial metabolic derangements are one of the risk factors of cardiovascular disease in humans. Insulin resistance and/or impaired early-phase insulin secretion are major determinants of postprandial metabolic derangements. In this study, we investigated the potential utility of combination therapy with vildagliptin, a dipeptidyl peptidase IV (DPP-IV) inhibitor and nateglinide, a rapid-onset/short-duration insulinotropic agent, for the treatment of postprandial metabolic derangements in Zucker Fatty (ZF) rats, an animal model of obesity with insulin resistance. ZF rats fed twice daily with or without high fat diet (HFD) were given vehicle, 50 mg/kg of nateglinide, 10 mg/kg of vildagliptin, or both for 6 weeks. Combination therapy with nateglinide and vildagliptin for 2 weeks ameliorated postprandial hyperglycemia, hypertriglyceridemia, and elevation of free fatty acid in ZF rats fed with HFD. 6-week treatment with nateglinide and vildagliptin not only increased hepatic levels of phosphorylated forkhead box protein 1A (FOXO1A), but also reduced triglyceride contents in the liver. Combination therapy also prevented the loss of pancreatic islet mass in ZF rats fed with HFD. These observations demonstrate that combination therapy with nateglinide and vildagliptin may improve postprandial metabolic derangements probably by ameliorating early phase of insulin secretion and hepatic insulin resistance, respectively, in ZF rats fed with HFD. Since combination therapy with nateglinide and vildagliptin restored the decrease in pancreatic beta cell mass, our present findings suggest that combination therapy could be a promising therapeutic strategy for postprandial dysmetabolism associated with obese and insulin resistance.

Positive association of serum levels of pigment epithelium-derived factor with high-sensitivity C-reactive protein in apparently healthy unmedicated subjects.

Pigment epithelium-derived factor (PEDF), a glycoprotein with neuronal differentiating activity, possesses antiinflammatory properties both in cell culture and animal models. However, the relationship between serum levels of PEDF and high-sensitivity C-reactive protein (hs-CRP), one of the representative biomarkers for inflammation in humans, is largely unknown. This study investigated whether serum PEDF levels were associated with hs-CRP in 120 apparently healthy unmedicated Japanese subjects (93 males, 27 females; mean age 58.0 years). All subjects underwent a complete history and physical examination, including blood chemistries, anthropometric and metabolic variables. Multiple regression analysis found that serum hs-CRP, creatinine and triglyceride levels, and the homeostasis model assessment of insulin resistance (HOMA-IR) and body mass index were significantly and independently associated with serum PEDF levels. It is concluded that, serum levels of PEDF are associated with serum levels of hs-CRP independently of anthropometric, metabolic and renal function variables. The results also suggest that serum PEDF levels may be elevated as a counter-system against subclinical inflammation.

Glyceraldehyde-derived advanced glycation end products (AGEs). A novel biomarker of postprandial hyperglycaemia in diabetic rats.

There is a growing body of evidence that postprandial hyperglycaemia plays an important role in accelerated atherosclerosis and may be a therapeutic target for preventing cardiovascular disease (CVD) in diabetes. However, there is no convenient biomarker that could reflect cumulative postprandial hyperglycaemia in diabetes. We have recently found that glyceraldehyde can rapidly react with amino groups of proteins to form glyceraldehyde-derived advanced glycation end products (AGEs), which evoke vascular inflammation and endothelial dysfunction, thereby being implicated in accelerated atherosclerosis in diabetes. In this study, we examined whether glyceraldehyde-derived AGEs were a biomarker that could reflect cumulative postprandial hyperglycaemia in Goto-Kakizaki (GK) rats fed twice a day. GK rats at 8 weeks of age were divided into 2 groups; either the vehicle (VEH) or 50 mg/kg of nateglinide (NAT) was administered twice daily just before each meal. After 6 weeks, nateglinide treatment was found to not only prevent postprandial hyperglycaemia, but also reduce glyceraldehyde-derived AGE levels in GK rats fed twice a day. However, there was no significant difference in HbA1c or glucose-derived AGE levels between the two groups. The present study demonstrated for the first time that glyceraldehyde-derived AGEs, but not HbA1c or glucose-derived AGEs, were a biomarker that could reflect cumulative postprandial hyperglycaemia in diabetic rats. Glyceraldehydederived AGEs may be a novel therapeutic target for preventing CVD in diabetes.