The role of Raf-1 kinase in diabetic retinopathy

Many metabolic pathways, including oxidative stress, PKC and the polyol pathway have been implicated in the development of diabetic retinopathy, but despite extensive research, its pathogenesis remains unclear. Recent studies have shown the role of a low-molecular-weight GTP-binding protein (H-Ras)-mediated signaling pathway in its development. The key effector protein of Ras function is a threonine/serine kinase-Raf kinase, and this kinase is involved in a variety of functions, including the cell cycle and proliferation and apoptosis. In animal models of diabetic retinopathy, Raf kinase is activated in the retina and its microvasculature. Activated Raf kinase is associated with increased apoptosis of retinal capillary cells, the process that precedes the development of retinal histopathology, and inhibition of Raf kinase ameliorates apoptosis. In clinical settings, inhibitors of Raf kinase have shown promising results in cancer treatment, and Raf kinase antisense oligonucleotides, iCo 007, is now in Phase II trial for macular edema, a chronic ocular disease associated with retinal neovascularization. Further elucidating the role of Raf kinase in diabetic retinopathy, and advances in the generation of antisense therapy for chronic diseases, should help test Raf antisense oligonucleotides for the treatment of this blinding complication that diabetic patients fear the most.     

Regulatory roles for Tiam1, a guanine nucleotide exchange factor for Rac1, in glucose-stimulated insulin secretion in pancreatic β-cells

Using various biochemical, pharmacological and molecular biological approaches, we have recently reported regulatory roles for Rac1, a small G-protein, in glucose-stimulated insulin secretion (GSIS). However, little is understood with respect to localization of, and regulation by, specific regulatory factors of Rac1 in GSIS. Herein, we investigated regulatory roles for Tiam1, a specific nucleotide exchange factor (GEF) for Rac1, in GSIS in pancreatic β-cells. Western blot analysis indicated that Tiam1 is predominantly cytosolic in distribution. NSC23766, a specific inhibitor of Tiam1-mediated activation of Rac1, markedly attenuated glucose-induced, but not KCl-induced insulin secretion in INS 832/13 cells and normal rat islets. Further, NSC23766 significantly reduced glucose-induced activation (i.e. GTP-bound form) and membrane association of Rac1 in INS 832/13 cells and rat islets. Moreover, siRNA-mediated knock-down of Tiam1 markedly inhibited glucose-induced membrane trafficking and activation of Rac1 in INS 832/13 cells. Interestingly, however, in contrast to the inhibitory effects of NSC23766, Tiam1 gene depletion potentiated GSIS in these cells; such a potentiation of GSIS was sensitive to extracellular calcium. Together, our studies present the first evidence for a regulatory role for Tiam1/Rac1-sensitive signaling step in GSIS. They also provide evidence for the existence of a potential Rac1/Tiam1-independent, but calcium-sensitive component for GSIS in these cells.     

The stimulus-secretion coupling of glucose-induced insulin release XLVI. Physiological role of l-glutamine as a fuel for pancreatic islets

Exogenous L-glutamine is actively metabolized in rat pancreatic islets. The rate of L-glutamine deamidation largely exceeds the rate of glutamate conversion to γ-aminobutyrate and α-ketoglutarate. The latter conversion occurs in part by oxidative deamination, and in part by transamination reactions coupled with the conversion of 2-keto acids (pyruvate, oxaloacetate), themselves derived from the metabolism of glutamine, to their corresponding amino acids (alanine, aspartate). An important fraction of malate formed from α-ketoglutarate leaves the Krebs cycle and is converted to pyruvate, this process being apparently associated with the induction of a more reduced state in cytosolic redox couples. L-Glutamine abolishes the oxidation of endogenous fatty acids and stimulates lipogenesis. A sparing action of L-glutamine upon the utilization of endogenous nutrients is documented by the fact that the glutamine-induced increase in O₂ consumption is much lower than expected from the rate of ¹⁴CO₂ output from islets exposed to L-[U-¹⁴C]glutamine. L-Glutamine, although decreasing K⁺conductance, fails to stimulate insulin release both in the absence and presence of D-glucose. It is proposed that L-glutamine represents a major fuel for pancreatic islets under physiological conditions.     

Retinal metabolic abnormalities in diabetic mouse: comparison with diabetic rat

PURPOSE: Dogs and rats are commonly used to examine the pathogenesis of diabetic retinopathy, but mouse is sparingly studied as an animal model of diabetic retinopathy. In this study metabolic abnormalities, postulated to contribute to the development of retinopathy in diabetes, are investigated in the retina of mice diabetic or galactose-fed for 2 months, and are compared to those obtained from hyperglycemic rats. METHODS: Diabetes was induced in mice (C57BL/6) and rats (Sprague Dawley) by alloxan injection, and experimental galactosemia by feeding normal animals diets supplemented with 30% galactose. After 2 months of hyperglycemia, levels of lipid peroxides, glutathione, nitric oxides and sorbitol, and activities of protein kinase C and (Na-K)-ATPase were measured in the retina. RESULTS: Two months of diabetes or experimental galactosemia in mice increased retinal oxidative stress, PKC activity and nitric oxides by 40-50% and sorbitol levels by 3 folds, and these abnormalities were similar to those observed in the retina of rats hyperglycemic for 2 months. CONCLUSIONS: Metabolic abnormalities, which are postulated to play important role in the development of diabetic retinopathy in other animal models, are present in the retina of diabetic mice, and the level of metabolic abnormalities is very comparable between mice and rats. Thus, mouse seems to be a promising animal model to study the pathogenesis of diabetic retinopathy.     

Studies on gastrointestinal tract functional changes in diabetic animals

Diarrhea and constipation are frequently reported gastrointestinal complications in diabetic patients. These disorders seem to be the consequence of altered innervations of a receptor system in the gastrointestinal tract in diabetes. We investigated the functional changes of cholinergic and beta-adrenergic receptor activities in rat ileum tissue after 8 weeks of control and streptozotocin (STZ)-induced diabetes in rats. Functional changes with different agonists were observed in diabetes: In diabetic rat ileum, carbachol increased the maximal contraction without changing pD2 values, whereas with acetylcholine, no changes were observed in the maximal contractile (Emax) response and pD2 values in diabetes were comparable with controls. With the beta-adrenoceptor agonists isoproterenol and salmeterol, decreased maximal relaxation responses were observed in 8-week diabetic rat ileums as compared with age-matched controls. With isoproterenol, pD2 values were also decreased in diabetic ileum, although not with salmeterol. On the other hand, with the in vivo charcoal meal test, the percentage of transit was increased in diabetic mice. The antitransit percentage was significantly increased in diabetic mice with the muscarinic antagonist atropine and the beta 2-selective agonist salbutamol, as compared with age-matched control mice. Results obtained from these in vivo and in vitro studies indicate that gastrointestinal complications such as diabetic diarrhea may occur because of functional changes such as increased intestinal transit and decreased intestinal tone due to increased cholinergic and decreased beta-adrenergic receptor activities in diabetes mellitus.     

New beyerane and isopimarane diterpenoids from Rhizophora mucronata

Chemical examination of the ethyl acetate extract of the roots of Rhizophora mucronata collected from Mangalore Coast resulted in the isolation of three more new diterpenoids, rhizophorins C-E (1-3) in addition to the two, rhizophorin A, (6R,11S,13S)-6,11,13-trihydroxy-2,3-seco-14-labden-2,8-olid-3-oic acid, and rhizophorin B, ent-3beta,20-epoxy-3alpha,18-dihydroxy-15-beyerene, recently reported. The structures of the new diterpenoids have been elucidated by a study of their physical and spectral data as 17-hydroxybeyer-15-en-3-one (1), 3beta,17-diacetoxy-15-beyeren-2-one (2) and 3beta,6alpha-diacetoxy-8(14),15-isopimaradien-2-one (3).     

Inhibitors of post-translational modifications of G-proteins as probes to study the pancreatic beta cell function: potential therapeutic implications

It is well established that glucose-induced insulin secretion involves generation of intracellular second messengers. Using specific inhibitors of guanosine triphosphate [GTP] biosynthesis [e.g., mycophenolic acid; MPA], we have identified a permissive role for GTP in glucose-stimulated insulin secretion. While the exact site of action for GTP within the islet beta cell remains to be identified and defined, recent evidence from several laboratories, including our own, indicate that it could involve activation of GTP-binding proteins [G-proteins]. These studies have identified both trimeric and monomeric forms of G-proteins within the pancreatic beta cell. Recent data also indicate that these G-proteins, specifically the monomeric G-proteins and the gamma subunits of trimeric G-proteins undergo a series of posttranslational modifications at their C-terminal cysteine. Such modifications include, isoprenylation, carboxyl methylation and palmitoylation. These modification steps appear to be essential for translocation of these proteins to the membrane sites for interaction with their respective effector proteins. This review primarily focuses on recent findings that clearly support the viewpoint that these posttranslational modification steps not only play obligatory roles in fuel-induced insulin secretion, but also in cytokine-mediated apoptotic demise of the beta cell. In this review, we also attempted to describe those findings involving the use of specific inhibitors for each of these pathways, and it is our hope that these aspects of beta cell metabolism and function generate interest in development of therapeutic intervention modalities to states of perturbed insulin release.     

Horiolide,a novel norditerpenoid from Indian ocean soft coral of the genus Sinularia

A novel norditerpenoid, horiolide (1), has been isolated from an Indian Ocean soft coral of the genus Sinularia. The structural elucidation was achieved by a study of its spectral characteristics. This compound is structurally characterized by a new carbon skeleton having one six-membered cyclohexane ring bearing an isopropylene moiety, a carbonyl group, and one seven-membered ring attached to a five-membered lactone moiety.     

Effect of long-term administration of alpha-lipoic acid on retinal capillary cell death and the development of retinopathy in diabetic rats

Oxidative stress is increased in the retina in diabetes, and it is considered to play an important role in the development of retinopathy. alpha-Lipoic acid, a thiol antioxidant, has been shown to have beneficial effects on polyneuropathy and on the parameters of oxidative stress in various tissues, including nerve, kidney, and retina. The purpose of this study was to examine the effect of alpha-lipoic acid on retinal capillary cell apoptosis and the development of pathology in diabetes. Retina was used from streptozotocin-induced diabetic rats receiving diets supplemented with or without alpha-lipoic acid (400 mg/kg) for 11 months of diabetes. Capillary cell apoptosis (by terminal transferase-mediated dUTP nick-end labeling) and formation of acellular capillaries were investigated in the trypsin-digested retinal microvessels. The effect of alpha-lipoic acid administration on retinal 8-hydroxy-2'deoxyguanosine (8-OHdG) and nitrotyrosine levels was determined by enzyme-linked immunosorbent assay. alpha-Lipoic acid administration for the entire duration of diabetes inhibited capillary cell apoptosis and the number of acellular capillaries in the retina, despite similar severity of hyperglycemia in the two diabetic groups (with and without alpha-lipoic acid). Retinal 8-OHdG and nitrotyrosine levels were increased by over twofold and 70%, respectively, in diabetes, and alpha-lipoic acid administration inhibited these increases. Our results demonstrate that the long-term administration of alpha-lipoic acid has beneficial effects on the development of diabetic retinopathy via inhibition of accumulation of oxidatively modified DNA and nitrotyrosine in the retina. alpha-Lipoic acid supplementation represents an achievable adjunct therapy to help prevent vision loss in diabetic patients.     

A rare prostaglandin from the soft coral Sarcophyton crassocaule of the Indian Ocean

The rare prostaglandin methyl (5Z)-9,15-dioxoprosta-5, 8(12)-dien-1-oate (1), hitherto unreported as a natural product, has been isolated from the Indian Ocean soft coral Sarcophyton crassocaule. Its structure was elucidated using detailed spectral ((1)H and (13)C NMR, DEPT, H-H COSY, C-H COSY, HRMS, and HMBC) analysis.