In vivo effects of vanadium on protein kinase B and key gluconeogenic enzymes in animal models of diabetes : comparison with insulin

Insulin resistance is a major characteristic of both Type 1 and Type 2 diabetes. However, despite numerous studies during the past decade the mechanism of insulin resistance is still not clear. Protein kinase B (PKB) has been proposed to be an intermediate protein kinase in the insulin signaling pathway by which insulin controls glucose disposal via stimulation of glycogen synthesis and glucose uptake in insulin sensitive tissues as well as hepatic glucose output. Hence, PKB may play a potential role in the development of insulin resistance. Vanadium compounds are known to have insulin mimetic/enhancing effects both in vitro and in vivo and therefore, are candidates for oral therapy in diabetes. Bis(maltolato)oxovanadium (IV) (BMOV) is an organic vanadium compound which corrects hyperglycemia in streptozotocin (STZ)-diabetic rats and lowers the elevated plasma insulin levels in fatty Zucker rats. In this study, we investigated 1) the association between PKB activity and insulin resistance 2) the in vivo effects of insulin and chronic BMOV treatment on PKB activity in the skeletal muscle and liver of two animal models of diabetes: STZ-diabetic Wistar rats, an animal model of poorly controlled Type 1 diabetes and fatty Zucker rats, an animal model that represents several characteristics of Type 2 diabetes. Animals were treated with BMOV in the drinking water (0.75-1 mg/ml) for 3 (or 8) weeks and sacrificed with or without insulin injection. Insulin (5 U/kg, i.v.) increased PKBα activity more than 10-fold and PKBβ activity more than 3-fold in both animal models. Despite the development of insulin resistance, PKBα activity was not impaired in STZ-diabetic rats up to 9 weeks of diabetes, thus excluding a role for PKBα in the development of insulin resistance in Type 1 diabetes. In contrast, insulin-induced PKBα (but not PKBβ) activity was markedly reduced in the skeletal muscle (fatty: 7-fold vs. lean: 14-fold), and significantly increased in the liver (fatty: 15.7-fold vs. lean: 7.6-fold) of fatty Zucker rats. These observations indicated an association between altered insulin-stimulated PKB activity and insulin resistance in this model. Comparison of basal PKBα activity in Zucker fatty and Zucker diabetic fatty rats indicated that basal enzyme activity was not affected by diabetes. BMOV, at doses sufficient to normalize fasting plasma glucose in STZ-diabetic rats and decrease plasma insulin levels in fatty Zucker rats had no detectable effect on basal or insulin-induced PKBα or PKBβ activities, indicating that the glucoregulatory effects of BMOV are independent of PKB activity in vivo. These findings led to the notion that BMOV may have more selective targets in the metabolic pathways. One potential pathway could be gluconeogenesis in the liver, since elevated hepatic glucose output is shown to be responsible for the fasting hyperglycemia in both types of diabetes. Hence, we tested the hypothesis that in vivo effects of vanadium may be mediated by changes in key gluconeogenic enzymes and inhibition of hepatic glucose output. STZ-diabetic rats were treated with BMOV in the drinking water (0.75-1 mg/ml) for 4 weeks or, for comparison, with insulin implants (4 U/day) for the final week of study. Phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), two key gluconeogenic enzymes, were measured in the liver and kidney, the main sites of endogenous glucose production. Treatment of STZ-diabetic rats with BMOV led to normalization of PEPCK enzyme activity and mRNA, and G6Pase mRNA levels in both liver and kidney, indicating that hypoglycemic effects of BMOV in STZ-diabetic rats are at least partially mediated by its direct and/or indirect effects on PEPCK and G6Pase mRNA expression. BMOV had no detectable effect on the expression of PEPCK and G6Pase in either liver or kidney in the non-diabetic rats. Furthermore, insulin treatment of STZ-diabetic rats restored the elevated mRNA levels of PEPCK and G6Pase in both tissues. In summary, results of this study demonstrated that: 1) In STZ-diabetic rats, both basal and insulin-stimulated PKBα activity were normal up to 9 weeks of diabetes, thus excluding a role for PKB in the development of insulin resistance in Type 1 diabetes. 2) In fatty Zucker rats, insulin-induced activation of PKBα (but not PKBβ) was markedly altered in the skeletal muscle and liver, indicating an association between PKBα activity and insulin resistance in this model. 3) Changes in PKBα activity were tissue specific implying that different mechanisms may be involved in the regulation of PKB. 4) The hypoglycemic effects of BMOV were at least partially mediated by its direct and/or indirect effects on PEPCK and G6Pase mRNA levels in STZ-diabetic rats via a PKB independent pathway.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

Mechanisms by which bis(maltolato)oxovanadium(IV) normalizes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression in streptozotocin-diabetic rats in vivo

Vanadium treatment normalizes plasma glucose levels in streptozotocin-diabetic rats in vivo, but the mechanism(s) involved are still unclear. Here, we tested the hypothesis that the in vivo effects of vanadium are mediated by changes in gluconeogenesis. Diabetic rats were treated with bis(maltolato)oxovanadium(IV) (BMOV) in the drinking water (0.75-1 mg/ml, 4 wk) or, for comparison, with insulin implants (4 U/d) for the final week of study. As with insulin, BMOV lowered plasma glucose and normalized phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) mRNA in the liver and kidney of diabetic rats. To determine the importance of reducing hyperglycemia per se, diabetic rats were treated either with a single ED dose of BMOV (0.1 mmol/kg, ip) or with phlorizin (900 mg/kg·d, 5 d). BMOV rapidly restored PEPCK and G-6-Pase mRNA and normalized plasma glucose in responsive (50%) diabetic rats but had no effect on the nonresponsive hyperglycemic rats. Phlorizin corrected plasma glucose but had no effect on PEPCK mRNA and only partially normalized G-6-Pase mRNA. In conclusion, 1) BMOV inhibits PEPCK mRNA expression and activity by rapid mechanisms that are not reproduced simply by correction of hyperglycemia; and 2) BMOV inhibits G-6-Pase expression by complex mechanisms that depend, in part, on correction of hyperglycemia. 5

Impaired NH2-terminal processing of human proislet amyloid polypeptide by the prohormone convertase PC2 leads to amyloid formation and cell death

Islet amyloid, formed by aggregation of islet amyloid polypeptide (IAPP; amylin), is a pathological characteristic of the pancreas in type 2 diabetes and may contribute to the progressive loss of beta-cells in this disease. We tested the hypothesis that impaired processing of the IAPP precursor proIAPP contributes to amyloid formation and cell death. GH3 cells lacking the prohormone convertase 1/3 (PC1/3) and IAPP and with very low levels of prohormone convertase 2 (PC2) were transduced with adenovirus (Ad) expressing human or rat (control) proIAPP linked to green fluorescent protein, with or without Ad-PC2 or Ad-PC1/3. Expression of human proIAPP increased the number of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells 96 h after transduction (+hIAPP 8.7 +/- 0.4% vs. control 3.0 +/- 0.4%; P < 0.05). COOH-terminal processing of human proIAPP by PC1/3 increased (hIAPP+PC1/3 10.4 +/- 0.7%; P < 0.05), whereas NH(2)-terminal processing of proIAPP by addition of PC2 markedly decreased (hIAPP+PC2 5.5 +/- 0.5%; P < 0.05) the number of apoptotic GH3 cells. Islets from mice lacking PC2 and with beta-cell expression of human proIAPP (hIAPP(+/+)/PC2(-/-)) developed amyloid associated with beta-cell death during 2-week culture. Rescue of PC2 expression by ex vivo transduction with Ad-PC2 restored NH(2)-terminal processing to mature IAPP and decreased both the extent of amyloid formation and the number of TUNEL-positive cells (-PC2 26.5 +/- 4.1% vs. +PC2 16.1 +/- 4.3%; P < 0.05). These findings suggest that impaired NH(2)-terminal processing of proIAPP leads to amyloid formation and cell death and that accumulation of the NH(2)-terminally extended human proIAPP intermediate may be a critical initiating step in amyloid formation

Amyloid formation disrupts the balance between interleukin-1β and interleukin-1 receptor antagonist in human islets

Objectives: β-cell dysfunction and apoptosis associated with islet inflammation play a key role in the pathogenesis of type 2 diabetes (T2D). Growing evidence suggests that islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to islet inflammation and β-cell death in T2D. We recently showed the role of interleukin-1β (IL-1β)/Fas/caspase-8 apoptotic pathway in amyloid-induced β-cell death. In this study, we used human islets in culture as an ex vivo model of amyloid formation to: (1) investigate the effects of amyloid on islet levels of the natural IL-1 receptor antagonist (IL-1Ra); (2) examine if modulating the IL-1β/IL-1Ra balance can prevent amyloid-induced β-cell Fas upregulation and apoptosis. Methods: Isolated human islets (n = 10 donors) were cultured in elevated glucose (to form amyloid) with or without a neutralizing human IL-1β antibody for up to 7 days. Parallel studies were performed with human islets in which amyloid formation was prevented by adeno-siRNA-mediated suppression of hIAPP expression (as control). β-cell levels of IL-1Ra, Fas, apoptosis as well as islet function, insulin- and amyloid-positive areas, and IL-1Ra release were assessed. Results: Progressive amyloid formation in human islets during culture was associated with alterations in IL-1Ra. Islet IL-1Ra levels were higher at early stages but were markedly reduced at later stages of amyloid formation. Furthermore, IL-1Ra release from human islets was reduced during 7-day culture in a time-dependent manner. These changes in IL-1Ra production and release from human islets during amyloid formation adversely correlated with islet IL-1β levels, β-cell Fas expression and apoptosis. Treatment with IL-1β neutralizing antibody markedly reduced amyloid-induced β-cell Fas expression and apoptosis, thereby improving islet β-cell survival and function during culture. Conclusions: These data suggest that amyloid formation impairs the balance between IL-1β and IL-1Ra in islets by increasing IL-1β production and reducing IL-1Ra levels thereby promoting β-cell dysfunction and death. Restoring the IL-1β/IL-1Ra ratio may provide an effective strategy to protect islet β-cells from amyloid toxicity in T2D

Role of beta-cell prohormone convertase (PC)1/3 in processing of pro-islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) (amylin), the major component of islet amyloid, is produced by cleavage at the COOH- and NH(2)-termini of its precursor, proIAPP, likely by the beta-cell prohormone convertases (PC) 1/3 and PC2. Mice lacking PC2 can process proIAPP at its COOH- but not its NH(2)-terminal cleavage site, suggesting that PC1/3 is capable of initiating proIAPP cleavage at its COOH-terminus. To determine the precise role of PC1/3 in proIAPP processing, Western blot analysis was performed on islets isolated from mice lacking PC1/3 (PC1/3(-/-)). These islets contained not only fully processed IAPP as in PC1/3(+/+) islets, but also elevated levels of a COOH-terminally unprocessed intermediate form, suggesting impaired processing at the COOH-terminus. Next, GH3 cells that do not normally express proIAPP or detectable levels of PC1/3 or PC2 were cotransduced with adenoviruses expressing rat proIAPP and either PC2 or PC1/3. As expected, in GH3 cells transduced to express only proIAPP, no processing was observed. Coexpression of proIAPP and PC2 resulted in production of mature IAPP, whereas in cells that coexpressed proIAPP and PC1/3 only a 6-kDa intermediate was produced. We conclude that PC1/3 is important for processing of proIAPP at the COOH-terminus, but in its absence, PC2 can initiate complete processing of proIAPP to IAPP by cleaving the precursor at either its NH(2)- or COOH-terminal cleavage sites

Impacts of high resolution model downscaling in coastal regions

The issue of appropriate resolution of coastal models is addressed in this paper. The quality of coastal predictions from three different spatial resolutions of a coastal ocean model is assessed in the context of simulation of the freshwater front in Liverpool Bay. Model performance is examined during the study period February 2008 using a 3-D baroclinic hydrodynamic model. Some characteristic lengthscales and non-dimensional numbers are introduced to describe the coastal plume and freshwater front. Metrics based on these lengthscales and the governing physical processes are used to assess model performance and these metrics have been calculated for the suite of downscaled models and compared with observations. Increased model resolution was found to better capture the position and strength of the freshwater front. However, instabilities along the front such as the tidal excursion led to large temporal and spatial variability in its position in the highest resolution model. By examining the spatial structure of the baroclinic Rossby radius in each model we identify which lengthscales are being resolved at different resolutions. In this dynamic environment it is more valuable to represent the governing time and space scales, rather than relying on strict point by point tests when evaluating model skill

Amyloid formation reduces protein kinase B phosphorylation in primary islet β-cells which is improved by blocking IL-1β signaling

<div><p>Amyloid formation in the pancreatic islets due to aggregation of human islet amyloid polypeptide (hIAPP) contributes to reduced β-cell mass and function in type 2 diabetes (T2D) and islet transplantation. Protein kinase B (PKB) signaling plays a key role in the regulation of β-cell survival, function and proliferation. In this study, we used human and hIAPP-expressing transgenic mouse islets in culture as two <i>ex vivo</i> models of human islet amyloid formation to: 1. Investigate the effects of amyloid formation on PKB phosphorylation in primary islet β-cells; 2. Test if inhibition of amyloid formation and/or interleukin-1β (IL-1β) signaling in islets can restore the changes in β-cell phospho-PKB levels mediated by amyloid formation. Human and hIAPP-expressing mouse islets were cultured in elevated glucose with an amyloid inhibitor (Congo red) or embedded within collagen matrix to prevent amyloid formation. To block the IL-1β signaling, human islets were treated with an IL-1 receptor antagonist (anakinra) or a glucagon-like peptide-1 agonist (exenatide). β-cell phospho-PKB levels, proliferation, apoptosis, islet IL-1β levels and amyloid formation were assessed. Amyloid formation in both cultured human and hIAPP-expressing mouse islets reduced β-cell phospho-PKB levels and increased islet IL-1β levels, both of which were restored by prevention of amyloid formation either by the amyloid inhibitor or embedding islets in collagen matrix, resulting in improved β-cell survival. Furthermore, inhibition of IL-1β signaling by treatment with anakinra or exenatide increased β-cell phospho-PKB levels, enhanced proliferation and reduced apoptosis in amyloid forming human islets during 7-day culture. These data suggest that amyloid formation leads to reduced PKB phosphorylation in β-cells which is associated with elevated islet IL-1β levels. Inhibitors of amyloid or amyloid-induced IL-1β production may provide a new approach to restore phospho-PKB levels thereby enhance β-cell survival and proliferation in conditions associated with islet amyloid formation such as T2D and clinical islet transplantation.</p></div

Treatment with anakinra or exenatide reduces IL-1β levels in human islets during culture which is associated with increased β-cell phospho-PKB levels, enhanced proliferation and reduced apoptosis.

<p>Islet sections from pre-culture and 7-day cultured human islets with anakinra (An; 10 μg/mL) or exenatide (Ex; 10 nmol/L) were immunolabelled for <b>(A)</b> insulin/IL-1β/Thio S or <b>(B)</b> insulin/p-PKB. <b>(C)</b> The percentage of islet amyloid area to total islet area in each condition. <b>(D)</b> β-cell phospho-PKB immunofluorescence (IF) intensity. The percentage of <b>(E)</b> PCNA-positive (proliferative) β-cells and <b>(F)</b> TUNEL-positive (apoptotic) β-cells. <b>(G)</b> Islet IL-1β release from 4-day cultured exenatide-treated and non-treated human islets. The arrows point to regions corresponding to enlarged areas in each image (original magnification: X400; insert: X1000). Results are expressed as mean +/- SEM of five independent studies (5 donors; 25–30 islets per condition in each study). For β-cell phospho-PKB IF intensity, quantifications were performed on a total of 20 amyloid-positive 7-day cultured human islets and equal number of anakinra- or exenatide-treated human islets (lower amyloid formation).*vs Day 0; <b>#</b>vs corresponding untreated group (<i>P<0</i>.<i>05</i>; one-way ANOVA or Student’s <i>t</i>-test).</p