James Lester Figarola

LR-90 a new advanced glycation endproduct inhibitor prevents progression of diabetic nephropathy in streptozotocin-diabetic rats

Aims/hypothesisAdvanced glycation and lipoxidation endproducts have been implicated in the pathogenesis of diabetic complications, including diabetic nephropathy. LR-90, a new advanced glycation endproduct inhibitor, was investigated for its effects on the development of renal disease in diabetic rats.MethodsDiabetic animals were randomly allocated into groups receiving LR-90 or vehicle (untreated). Age- and weight-matched non-diabetic rats were studied concurrently. Body weight, plasma glucose, glycated haemoglobin, urinary albumin and creatine excretions were measured serially. Kidney histopathology, AGE accumulation in cells and tissues, protein oxidation, were also examined. In vitro assays were used to assess the possible mechanism of action of LR-90.ResultsLR-90 inhibited the increase in albumin and creatinine concentrations, and concentrations of circulating AGE in diabetic rats without any effect on glycaemic control. LR-90 treated-rats also showed higher body weights than untreated diabetic rats. LR-90 prevented glomerulosclerosis, tubular degeneration and collagen deposition in the kidney. AGE-induced cross-linking and fluorescence of tail collagen were reduced by LR-90 treatment. LR-90 also decreased AGE accumulation in kidney glomeruli and nitrotyrosine deposition in the renal cortex. In vitro, LR-90 was capable of reacting with reactive carbonyl compounds and was a more potent metal chelator than pyridoxamine and aminoguanidine.Conclusion/interpretationLR-90 reduces in vivo AGE accumulation, AGE-protein cross-linking and protein oxidation, and could be beneficial in preventing the progression of diabetic nephropathy. The AGE inhibitory and therapeutic effects of LR-90 could be attributed, at least in part, to its ability to react with reactive carbonyl species and/or potent metal chelating activity that inhibits glycoxidative-AGE formation.

Anti-Inflammatory Effects of the Advanced Glycation End Product Inhibitor LR-90 in Human Monocytes

Ligation of advanced glycation end products (AGEs) with their receptor (RAGE) plays an important role in the development of various diabetes complications, including atherosclerosis. Monocyte activation, adhesion, and migration are key events in the pathogenesis of atherosclerosis. Previous studies showed that AGEs and S100b, a specific RAGE ligand, could augment monocyte inflammatory responses via RAGE. In this study, we examined whether LR-90, a compound belonging to a new class of AGE inhibitor, could inhibit inflammatory responses in human monocytes. Human THP-1 cells were pretreated with LR-90 and then stimulated with S100b. LR-90 significantly inhibited S100b-induced expression of RAGE and other proinflammatory genes including monocyte chemoattractant protein-1, interferon-γ–inducible protein-10, and cyclooxygenase-2 in a dose-dependent manner. These inhibitory effects may be exerted via inhibition of nuclear factor-κB (NF-κB) activation, as LR-90 suppressed both S100b–and tumor necrosis factor-α–induced IκB-α degradation as well as NF-κB promoter transcriptional activity. LR-90 also prevented oxidative stress in activated monocytes, as demonstrated by its inhibitory effects on S100b-induced expression of NADPH oxidase and intracellular superoxide production. In addition, LR-90 blocked S100b-induced monocyte adhesion to human umbilical vein endothelial cell. These new data show that, in addition to its AGE inhibitory effects, LR-90 has novel anti-inflammatory properties and might therefore have additional protective effects against diabetic vascular complications.

A new advanced glycation inhibitor, LR-90, prevents experimental diabetic retinopathy in rats

Background: Diabetic retinopathy is associated with accumulation of advanced glycation end products in the retinal microvasculature. LR-90 is an effective multistage inhibitor of advanced glycation with renoprotective and anti-inflammatory properties. Aim: To explore the role of LR-90 in the progression of experimental diabetic retinopathy. Methods: Streptozotocin-induced diabetic Sprague–Dawley rats were treated with LR-90 (50 mg/l in drinking water) for up to 32 weeks. At the end of the study, eyes were enucleated and subjected to trypsin digestion and staining with light green/haematoxylin. Acellular capillaries and pericytes were quantified in random fields using light microscopy. Results: In the LR-90-treated diabetic animals, acellular capillary numbers were reduced to 1.63 (0.20) from 2.58 (0.49) (p<0.05) in diabetic controls. LR-90 treatment also restored the pericyte deficit from 18.12 (0.98) in diabetic rats to 24.19 (0.76) (p<0.001). Conclusion: These findings show that LR-90 can effectively inhibit important lesions of diabetic retinopathy. This agent has potential for preventing retinopathy in patients with diabetes.

LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells.

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis.

Prevention of early renal disease, dyslipidaemia and lipid peroxidation in STZ-diabetic rats by LR-9 and LR-74, novel AGE inhibitors

Increased formation of advanced glycation/lipoxidation endproducts (AGEs/ALEs) has been implicated in the pathogenesis of various diabetic complications. Several compounds have been developed as inhibitors of AGE/ALE formation. We examined the effects of two new AGE/ALE inhibitors, LR‐9 and LR‐74, on the development of early renal disease and lipid metabolism in streptozotocin (STZ)‐induced diabetic rats.

COH-SR4 Reduces Body Weight, Improves Glycemic Control and Prevents Hepatic Steatosis in High Fat Diet- Induced Obese Mice

Obesity is a chronic metabolic disorder caused by imbalance between energy intake and expenditure, and is one of the principal causative factors in the development of metabolic syndrome, diabetes and cancer. COH-SR4 (“SR4”) is a novel investigational compound that has anti-cancer and anti-adipogenic properties. In this study, the effects of SR4 on metabolic alterations in high fat diet (HFD)-induced obese C57BL/J6 mice were investigated. Oral feeding of SR4 (5 mg/kg body weight.) in HFD mice for 6 weeks significantly reduced body weight, prevented hyperlipidemia and improved glycemic control without affecting food intake. These changes were associated with marked decreases in epididymal fat mass, adipocyte hypertrophy, increased plasma adiponectin and reduced leptin levels. SR4 treatment also decreased liver triglycerides, prevented hepatic steatosis, and normalized liver enzymes. Western blots demonstrated increased AMPK activation in liver and adipose tissues of SR4-treated HFD obese mice, while gene analyses by real time PCR showed COH-SR4 significantly suppressed the mRNA expression of lipogenic genes such as sterol regulatory element binding protein-1c (Srebf1), acetyl-Coenzyme A carboxylase (Acaca), peroxisome proliferator-activated receptor gamma (Pparg), fatty acid synthase (Fasn), stearoyl-Coenzyme A desaturase 1 (Scd1), carnitine palmitoyltransferase 1a (Cpt1a) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (Hmgcr), as well as gluconeogenic genes phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6pc) in the liver of obese mice. In vitro, SR4 activates AMPK independent of upstream kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). Together, these data suggest that SR4, a novel AMPK activator, may be a promising therapeutic compound for treatment of obesity, fatty liver disease, and related metabolic disorders.

Small‑molecule COH-SR4 inhibits adipocyte differentiation via AMPK activation.

Obesity is a chronic metabolic disorder caused by an imbalance between energy intake and expenditure. It is one of the principal causative factors involved in the development of metabolic syndrome and cancer. Inhibition of adipocyte differentiation has often been a target of anti-obesity strategies since obesity is caused not only by hypertrophy but also by adipocyte hyperplasia. In this study, we investigated the effects of COH-SR4, a novel compound with anticancer properties, on the adipogenesis in 3T3-L1 cells. Treatment with COH-SR4 significantly inhibited adipocyte differentiation in a dose-dependent manner. This inhibitory effect mainly occurred at the early phase of differentiation through inhibition of mitotic clonal expansion and cell cycle arrest at the G1/S phase transition. In differentiating adipocytes, COH-SR4 significantly reduced intracellular lipid accumulation and downregulated the expression of key adipogenesis-related transcription factors and lipogenic proteins. COH-SR4 exhibited no cytotoxic effects in 3T3-L1 cells, but indirectly activated AMP-activated protein kinase (AMPK). AMPK activation by COH-SR4 also resulted in the phosphorylation of raptor and tuberous sclerosis protein 2 (TSC2), two proteins involved in the mammalian target of rapamycin (mTOR) signaling pathways. Additionally, COH-SR4 decreased the phosphorylation of p70 kDa ribosomal protein S6 kinase (S6K) and initiation factor 4E (eIF4E) binding protein 1 (4EB‑P1), two downstream effectors of mTOR that regulate protein synthesis. Interestingly, knockdown of AMPKα1/α2 prevented the ability of COH-SR4 to inhibit cell cycle arrest and overall adipogenesis and lipid accumulation in the differentiating 3T3-L1 cells. Taken together, these results suggest that COH-SR4 inhibits 3T3-L1 adipogenesis via AMPK activation. COH-SR4 may be a promising compound for the treatment of obesity and related metabolic disorders.

Renoprotective and lipid-lowering effects of LR compounds, novel advanced glycation end product inhibitors, in streptozotocin-induced diabetic rats.

Abstract: The accelerated formation of advanced glycation/lipoxidation end products (AGEs/ALEs) has been implicated in the pathogenesis of various diabetic complications. Several natural and synthetic compounds have been proposed and advanced as inhibitors of AGE/ALE formation. We examined the effects of two new AGE/ALE inhibitors, LR‐9 and LR‐74, on the prevention of early renal disease and dyslipidemia in streptozotocin (STZ)‐induced diabetic rats. Diabetic rats were treated with either LR‐9 or LR‐74 for 32 weeks. Progression of renal disease was evaluated by measurements of urinary albumin and plasma creatinine concentrations. AGE‐induced chemical modification of the tail tendon collagen and levels of Nε‐(carboxymethyl)‐ and (carboxyethyl)‐ lysines (CML and CEL) in skin collagen were measured. AGE/ALE levels in kidneys were determined by immunohistochemistry. Plasma lipids and their lipid hydroperoxide concentrations were also determined. Treatment of either LR‐9 or LR‐74 significantly inhibited the increase in albuminuria, plasma creatinine, hyperlipidemia, and plasma lipid peroxidation in diabetic rats without any effects on hyperglycemia. Both compounds also reduced CML‐AGE accumulation in kidney glomeruli and tubules, AGE‐linked fluorescence and cross‐linking of tail collagen, and levels of CML and CEL in skin collagen. These results suggest that both LR compounds can inhibit the progression of renal disease and also prevent dyslipidemia in experimental diabetes. These compounds may have an additional beneficial effect as an antioxidant against lipid peroxidation, and thus may provide alternative therapeutic options for the treatment of various diabetic macrovascular complications.

RLIP76 Protein Knockdown Attenuates Obesity Due to a High-fat Diet

Background: RLIP76 homozygous knock-out mice (RLIP76−/−) display a partially anti-metabolic syndrome phenotype characterized by insulin sensitivity, hypoglycemia, and hypolipidemia. Results: RLIP76−/− mice are highly resistant to obesity as well as these other features of metabolic syndrome (MetS) caused by a high-fat diet. Conclusion: Our findings confirm a fundamental role of RLIP76 in regulating the function of obesity-promoting pro-inflammatory cytokines. Significance: Present studies provide a novel mechanism for targeted therapy of obesity and MetS. Feeding a Western high-fat diet (HFD) to C57BL/6 mice induces obesity, associated with a chronic inflammatory state, lipid transport, and metabolic derangements, and organ system effects that particularly prominent in the kidneys. Here, we report that RLIP76 homozygous knock-out (RLIP76−/−) mice are highly resistant to obesity as well as these other features of metabolic syndrome caused by HFD. The normal increase in pro-inflammatory and fibrotic markers associated with HFD induced obesity in wild-type C57B mice was broadly and nearly completely abrogated in RLIP76−/− mice. This is a particularly striking finding because chemical markers of oxidative stress including lipid hydroperoxides and alkenals were significantly higher in RLIP76−/− mice. Whereas HFD caused marked suppression of AMPK in wild-type C57B mice, RLIP76−/− mice had baseline activation of AMP-activated protein kinase, which was not further affected by HFD. The baseline renal function was reduced in RLIP76−/− mice as compared with wild-type, but was unaffected by HFD, in marked contrast to severe renal impairment and glomerulopathy in the wild-type mice given HFD. Our findings confirm a fundamental role of RLIP76 in regulating the function of obesity-promoting pro-inflammatory cytokines, and provide a novel mechanism for targeted therapy of obesity and metabolic syndrome.

RLIP76 Targeted Therapy for Kidney Cancer

ABSTRACTDespite recent improvements in chemotherapeutic approaches to treating kidney cancer, this malignancy remains deadly if not found and removed at an early stage of the disease. Kidney cancer is highly drug-resistant, which may at least partially result from high expression of transporter proteins in the cell membranes of kidney cells. Although these transporter proteins can contribute to drug-resistance, targeting proteins from the ATP-binding cassette transporter family has not been effective in reversing drug-resistance in kidney cancer. Recent studies have identified RLIP76 as a key stress-defense protein that protects normal cells from damage caused by stress conditions, including heat, ultra-violet light, X-irradiation, and oxidant/electrophilic toxic chemicals, and is crucial for protecting cancer cells from apoptosis. RLIP76 is the predominant glutathione-electrophile-conjugate (GS-E) transporter in cells, and inhibiting it with antibodies or through siRNA or antisense causes apoptosis in many cancer cell types. To date, blocking of RLIP76, either alone or in combination with chemotherapeutic drugs, as a therapeutic strategy for kidney cancer has not yet been evaluated in human clinical trials, although there is considerable potential for RLIP76 to be developed as a therapeutic agent for kidney cancer. In the present review, we discuss the mechanisms underlying apoptosis caused by RLIP76 depletion, the role of RLIP76 in clathrin-dependent endocytosis deficiency, and the feasibility of RLIP76-targeted therapy for kidney cancer.