Metabolic memory in diabetes – from in vitro oddity to in vivo problem: Role of Apoptosis

Retinal capillary cells undergo apoptosis before pathology characteristic of retinopathy can be observed, and the appearance of apoptotic capillary cell can predict the development of pathology. The purpose of this study is to investigate the effect of reversal of hyperglycemia on retinal capillary cell apoptosis, and identify the apoptosis encoding genes. Streptozotocin-diabetic rats were maintained either in poor glycemic control (PC, glycated hemoglobin, GHb >11%) or in good glycemic control (GC, GHb <6%) for 12 months, or allowed to be in PC for 6 months followed by GC for 6 additional months (PC–GC). Capillary cell apoptosis was determined in the trypsin-digested retinal microvasculature by TUNEL staining, and the genes encoding apoptosis were identified by Oligo GEArray rat apoptosis microarray that profiles 113 genes. Six months of good glycemic control that followed 6 months of poor control failed to attenuate the number of TUNEL-positive capillary cells in the retinal microvasculature. Twenty-three retinal genes, mainly from TNF ligand and receptor, caspase, Bcl-2 and death domain subfamilies that were upregulated by least a two-fold in PC rats remain upregulated after reversal of hyperglycemia. Thus, the continued activation of apoptosis plays a major role in the resistance of retinopathy to halt after re-institution of good glycemic control, and the regulation apoptosis machinery could help retard the progression of diabetic retinopathy.     

A defect late in stimulus-secretion coupling impairs insulin secretion in Goto-Kakizaki diabetic rats.

A widely accepted genetically determined rodent model for human type 2 diabetes is the Goto-Kakizaki (GK) rat; however, the lesion(s) in the pancreatic islets of these rats has not been identified. Herein, intact islets from GK rats (aged 8-14 weeks) were studied, both immediately after isolation and after 18 h in tissue culture. Despite intact contents of insulin and protein, GK islets had markedly deficient insulin release in response to glucose, as well as to pure mitochondrial fuels or a non-nutrient membrane-depolarizing stimulus (40 mmol/l K+). In contrast, mastoparan (which activates GTP-binding proteins [GBPs]) completely circumvented any secretory defect. Basal and stimulated levels of adenine and guanine nucleotides, the activation of phospholipase C by Ca2+ or glucose, the secretory response to pertussis toxin, and the activation of selected low-molecular weight GBPs were not impaired. Defects were found, however, in the autophosphorylation and catalytic activity of cytosolic nucleoside diphosphokinase (NDPK), which may provide compartmentalized GTP pools to activate G-proteins; a deficient content of phosphoinositides was also detected. These studies identify novel, heretofore unappreciated, defects late in signal transduction in the islets of our colony of GK rats, possibly occurring at the site of activation by NDPK of a mastoparan-sensitive G-protein-dependent step in exocytosis.     

Tiam1/Rac1 signaling pathway mediates palmitate-induced, ceramide-sensitive generation of superoxides and lipid peroxides and the loss of mitochondrial membrane potential in pancreatic β-cells

The phagocytic NADPH oxidase [NOX] has been implicated in the generation of superoxides in the pancreatic β-cell. Herein, using normal rat islets and clonal INS 832/13 cells, we tested the hypothesis that activation of the small G-protein Rac1, which is a member of the NOX holoenzyme, is necessary for palmitate [PA]-induced generation of superoxides in pancreatic β-cells. Incubation of isolated β-cells with PA potently increased the NOX activity culminating in a significant increase in the generation of superoxides and lipid peroxides in these cells; such effects of PA were attenuated by diphenyleneiodonium [DPI], a known inhibitor of NOX. In addition, PA caused a transient, but significant activation [i.e., GTP-bound form] of Rac1 in these cells. NSC23766, a selective inhibitor of Rac1, but not Cdc42 or Rho activation, inhibited Rac1 activation and the generation of superoxides and lipid peroxides induced by PA. Fumonisin B-1 [FB-1], which inhibits de novo synthesis of ceramide [CER] from PA, also attenuated PA-induced superoxide and lipid peroxide generation and NOX activity implicating intracellularly generated CER in the metabolic effects of PA; such effects were also demonstrable in the presence of the cell-permeable C2-CER. Further, NSC23766 prevented C2-CER-induced Rac1 activation and production of superoxides and lipid peroxides. Lastly, C2-CER, but not its inactive analogue, significantly reduced the mitochondrial membrane potential, which was prevented to a large degree by NSC23766. Together, our findings suggest that Tiam1/Rac1 signaling pathway regulates PA-induced, CER-dependent superoxide generation and mitochondrial dysfunction in pancreatic β-cells.     

Trocheliophorin: A novel rearranged sesquiterpenoid from the Indian Ocean soft coral Sarcophyton trocheliophorum

A novel rearranged sesquiterpenoid, trocheliophorin (1) has been isolated from the soft coral Sarcophyton trocheliophorum besides the known compounds, sarcophytin, methyl arachidonate, and two polyhydroxy steroids (24S)-24-methylcholestane-3beta,5alpha,6beta,25-tetrol-25-monoacetate and (24S)-24-methylcholestane-3beta,5alpha,6beta,25-tetrol. The structure of the new sesquiterpenoid was elucidated by a study of its spectral data.     

Glucose- and GTP-dependent stimulation of the carboxyl methylation of CDC42 in rodent and human pancreatic islets and pure beta cells. Evidence for an essential role of GTP-binding proteins in nutrient-induced insulin secretion.

Several GTP-binding proteins (G-proteins) undergo post-translational modifications (isoprenylation and carboxyl methylation) in pancreatic beta cells. Herein, two of these were identified as CDC42 and rap 1, using Western blotting and immunoprecipitation. Confocal microscopic data indicated that CDC42 is localized only in islet endocrine cells but not in acinar cells of the pancreas. CDC42 undergoes a guanine nucleotide-specific membrane association and carboxyl methylation in normal rat islets, human islets, and pure beta (HIT or INS-1) cells. GTPgammaS-dependent carboxyl methylation of a 23-kD protein was also demonstrable in secretory granule fractions from normal islets or beta cells. AFC (a specific inhibitor of prenyl-cysteine carboxyl methyl transferases) blocked the carboxyl methylation of CDC42 in five types of insulin-secreting cells, without blocking GTPgammaS-induced translocation, implying that methylation is a consequence (not a cause) of transfer to membrane sites. High glucose (but not a depolarizing concentration of K+) induced the carboxyl methylation of CDC42 in intact cells, as assessed after specific immunoprecipitation. This effect was abrogated by GTP depletion using mycophenolic acid and was restored upon GTP repletion by coprovision of guanosine. In contrast, although rap 1 was also carboxyl methylated, it was not translocated to the particulate fraction by GTPgammaS; furthermore, its methylation was also stimulated by 40 mM K+ (suggesting a role which is not specific to nutrient stimulation). AFC also impeded nutrient-induced (but not K+-induced) insulin secretion from islets and beta cells under static or perifusion conditions, whereas an inactive structural analogue of AFC failed to inhibit insulin release. These effects were reproduced not only by S-adenosylhomocysteine (another methylation inhibitor), but also by GTP depletion. Thus, the glucose- and GTP-dependent carboxyl methylation of G-proteins such as CDC42 is an obligate step in the stimulus-secretion coupling of nutrient-induced insulin secretion, but not in the exocytotic event itself. Furthermore, AFC blocked glucose-activated phosphoinositide turnover, which may provide a partial biochemical explanation for its effect on secretion, and implies that certain G-proteins must be carboxyl methylated for their interaction with signaling effector molecules, a step which can be regulated by intracellular availability of GTP.     

Depletion of the catalytic subunit of protein phosphatase-2A (PP2Ac) markedly attenuates glucose-stimulated insulin secretion in pancreatic beta-cells

Among various phosphatases, the protein phosphatase 2A (PP2A) is relatively well studied in the islet. Previously, we have demonstrated that the catalytic subunit of PP2A (PP2Ac) undergoes okadaic acid (OKA)-sensitive, reversible carboxylmethylation (CML), which appears to be requisite for glucose-stimulated insulin secretion (GSIS). Using the siRNA approach, we examined, herein, the contributory roles of PP2Ac in GSIS from insulin-secreting pancreatic β-(INS-1 832/13) cells. Immunologically, PP2Ac was detectable in all the subcellular fractions studied in rank order of: cytosol > microsomes > secretory granules = nucleus > mitochondria. Transfection of PP2Ac-specific, but not scrambled-siRNA, markedly attenuated PP2A activity and GSIS in these cells. Together, our findings provide a direct evidence for a positive modulatory role for PP2Ac in signaling steps leading to GSIS.     

Diabetic retinopathy, superoxide damage and antioxidants

Retinopathy, the leading cause of acquired blindness in young adults, is one of the most feared complications of diabetes, and hyperglycemia is considered as the major trigger for its development. The microvasculature of the retina is constantly bombarded by high glucose, and this insult results in many metabolic, structural and functional changes. Retinal mitochondria become dysfunctional, its DNA is damaged and proteins encoded by its DNA are decreased. The electron transport chain system becomes compromised, further producing superoxide and providing no relief to the retina from a continuous cycle of damage. Although the retina attempts to initiate repair mechanisms by inducing gene expressions of the repair enzymes, their mitochondrial accumulation remains deficient. Understanding the molecular mechanism of mitochondrial damage should help identify therapies to treat/retard this sight threatening complication of diabetes. Our hope is that if the retinal mitochondria are maintained healthy with adjunct therapies, the development and progression of diabetic retinopathy can be inhibited.     

An integrated WRF/HYSPLIT modeling approach for the assessment of PM2.5 source regions over the Mississippi Gulf Coast region

Fine particulate matter (PM_2.5) is majorly formed by precursor gases, such as sulfur dioxide (SO₂) and nitrogen oxides (NO_x), which are emitted largely from intense industrial operations and transportation activities. PM_2.5 has been shown to affect respiratory health in humans. Evaluation of source regions and assessment of emission source contributions in the Gulf Coast region of the USA will be useful for the development of PM_2.5 regulatory and mitigation strategies. In the present study, the Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) model driven by the Weather Research & Forecasting (WRF) model is used to identify the emission source locations and transportation trends. Meteorological observations as well as PM_2.5 sulfate and nitric acid concentrations were collected at two sites during the Mississippi Coastal Atmospheric Dispersion Study, a summer 2009 field experiment along the Mississippi Gulf Coast. Meteorological fields during the campaign were simulated using WRF with three nested domains of 36, 12, and 4?km horizontal resolutions and 43 vertical levels and validated with North American Mesoscale Analysis. The HYSPLIT model was integrated with meteorological fields derived from the WRF model to identify the source locations using backward trajectory analysis. The backward trajectories for a 24-h period were plotted at 1-h intervals starting from two observation locations to identify probable sources. The back trajectories distinctly indicated the sources to be in the direction between south and west, thus to have origin from local Mississippi, neighboring Louisiana state, and Gulf of Mexico. Out of the eight power plants located within the radius of 300?km of the two monitoring sites examined as sources, only Watson, Cajun, and Morrow power plants fall in the path of the derived back trajectories. Forward dispersions patterns computed using HYSPLIT were plotted from each of these source locations using the hourly mean emission concentrations as computed from past annual emission strength data to assess extent of their contribution. An assessment of the relative contributions from the eight sources reveal that only Cajun and Morrow power plants contribute to the observations at the Wiggins Airport to a certain extent while none of the eight power plants contribute to the observations at Harrison Central High School. As these observations represent a moderate event with daily average values of 5?C8???g?m^?3 for sulfate and 1?C3???g?m^?3 for HNO₃ with differences between the two spatially varied sites, the local sources may also be significant contributors for the observed values of PM_2.5.     

Evidence for phosphorylation of pancreatic islet pyruvate kinase

Rabbit pancreatic islet cytosol catalyzes the calcium-activated phosphorylation by [gamma 32P]ATP of a protein with a molecular weight of 57,000 that is precipitated with antipyruvate kinase antibodies. We were unable to demonstrate that phosphorylation in the presence of calcium or cAMP had any immediate effect on rat pancreatic islet pyruvate kinase activity. This finding is consistent with our inability to confirm the finding of others that pancreatic islets contain phosphoenolpyruvate carboxykinase activity (Diabetes, 34:246, 1985). Since the carboxykinase catalyzes phosphoenolpyruvate formation and pyruvate kinase catalyzes essentially the opposite reaction, if the carboxykinase were present in the beta cell, pyruvate kinase would need to be inhibited to prevent recycling of phosphoenolpyruvate.