A bioluminescent Pseudomonas aeruginosa wound model reveals increased mortality of type 1 diabetic mice to biofilm infection.

OBJECTIVE: To examine how bacterial biofilms, as contributing factors in the delayed closure of chronic wounds in patients with diabetes, affect the healing process. METHOD: We used daily microscopic imaging and the IVIS Spectrum in vivo imaging system to monitor biofilm infections of bioluminescent Pseudomonas aeruginosa and evaluate healing in non-diabetic and streptozotocin-induced diabetic mice. RESULTS: Our studies determined that diabetes alone did not affect the rate of healing of full-depth murine back wounds compared with non-diabetic mice. The application of mature biofilms to the wounds significantly decreased the rate of healing compared with non-infected wounds for both non-diabetic as well as diabetic mice. Diabetic mice were also more severely affected by biofilms displaying elevated pus production, higher mortality rates and statistically significant increase in wound depth, granulation/fibrosis and biofilm presence. Introduction of a mutant Pseudomonas aeruginosa capable of producing high concentrations of cyclic di-GMP did not result in increased persistence in either diabetic or non-diabetic animals compared with the wild type strain. CONCLUSION: Understanding the interplay between diabetes and biofilms may lead to novel treatments and better clinical management of chronic wounds.

Non-mammalian fat-1 gene prevents neoplasia when introduced to a mouse hepatocarcinogenesis model: Omega-3 fatty acids prevent liver neoplasia.

We investigated the effect of a non-mammalian omega-3 desaturase in a mouse hepatocarcinogenesis model. Mice containing double mutations (DM) in c-myc and TGF-alpha (transforming growth factor-alpha), leading to liver neoplasia, were crossed with mice containing omega-3 desaturase. MRI analysis of triple mutant (TM) mice showed the absence of neoplasia at all time points for 92% of mice in the study. Pathological changes of TM (TGFalpha/c-myc/fat-1) mouse liver tissue was similar to control mouse liver tissue. Magnetic resonance spectroscopy (MRS) measurements of unsaturated fatty acids found a significant difference (p<0.005) between DM and TM transgenic (Tg) mice at 34 and 40 weeks of age. HPLC analysis of mouse liver tissue revealed markedly decreased levels of omega-6 fatty acids in TM mice when compared to DM (TGFalpha/c-myc) and control (CD1) mice. Mass spectrometry (MS) analysis indicated significantly decreased 16:0/20:4 and 18:1/20:4 and elevated 16:0/22:6 fatty acyl groups in both GPCho and GPEtn, and elevated 16:0/20:5, 18:0/18:2, 18:0/18:1 and 18:0/22:6 in GPCho, within TM mice compared to DM mice. Total fatty acid analysis indicated a significant decrease in 18:1n9 in TM mice compared to DM mice. Western blot analysis of liver tissue showed a significant (p<0.05) decrease in NF-kappaB (nuclear factor-kappaB) levels at 40 weeks of age in TM mice compared to DM mice. Microarray analysis of TM versus DM mice livers at 40 weeks revealed alterations in genes involved in cell cycle regulation, cell-to-cell signaling, p53 signaling, and arachidonic acid (20:4) metabolism. Endogenous omega-3 fatty acids were found to prevent HCC development in mice.

Glucose-specific regulation of aldose reductase in capan-1 human pancreatic duct cells In vitro.

Impaired pancreatic duct secretion is frequently observed in insulin-dependent diabetes mellitus (IDDM), although the cellular mechanism(s) of dysfunction remains unknown. Studies in other tissues have suggested that a hyperglycemia-induced decrease in Na, K-ATPase activity could contribute to the metabolic complications of IDDM and that increased polyol metabolism is involved in this response. The present studies examined the effects of glucose on Na, K-ATPase activity and on expression and activity of aldose reductase (AR), a primary enzyme of polyol metabolism, in Capan-1 human pancreatic duct cells. Increasing medium glucose from 5.5 to 22 mM caused a 29% decrease in Na,K-ATPase activity. The decrease was corrected by 100 microM sorbinil, a specific AR inhibitor. Increasing glucose from 5.5 to 110 mM also resulted in concentration-dependent increases in AR mRNA and enzyme activity that could be resolved into two components, one that was glucose specific and observed at pathophysiological concentrations (< 55 mM) and a second that was osmotically induced at high concentrations (> 55 mM) and which was not glucose specific. The present study demonstrates that pathophysiological levels of glucose specifically activate polyol metabolism with a consequent decrease in Na,K-ATPase activity in pancreatic duct epithelial cells, and that this response to hyperglycemia could contribute to decreased pancreatic secretion observed in IDDM. This is the first report of AR regulation in the pancreatic duct epithelium.

Glucose-specific regulation of aldose reductase in human retinal pigment epithelial cells in vitro.

PURPOSE: To test the hypothesis that pathophysiological levels of glucose regulate aldose reductase (AR2) gene expression, protein production, and activity in human retinal pigment epithelial (RPE) cells in vitro. METHODS: Primary cultures of human RPE cells were grown for up to 72 hours in media supplemented with various concentrations of glucose (5, 20, or 75 mM), or in 5 mM glucose containing media supplemented with one of the following: galactose, the transported but nonmetabolized glucose analogue 3-O-methylglucose (3-OMG), or the impermeant hexitol mannitol-so that the final hexose concentrations were equimolar to those of the various glucose concentrations used. Changes in the transcript levels for AR2 mRNA, AR2 protein content, and AR2 enzyme activity were determined. RPE glucose utilization and lactate production were determined in media containing 5 and 20 mM glucose. RESULTS: Glucose utilization and lactate production increased 4.8-fold and 4.4-fold, respectively, when RPE cells were grown in media containing 20 mM versus 5 mM glucose. Glucose was more effective than any other hexose in the induction of AR2 mRNA or increased AR2 protein expression. When RPE cells were grown in media containing 20 mM mannitol, 3-OMG, or galactose they had lower levels of AR2 mRNA expression than when cells were grown in medium containing 5 mM glucose. RPE cells grown in medium supplemented with 20 or 75 mM galactose did not show a greater increase in AR2 protein expression than cells grown in medium containing 5 mM glucose. Hyperosmotic induction of AR2 mRNA was the same in medium containing 75 mM glucose or 75 mM mannitol, but was at least 50% lower when RPE cells were grown in 75 mM galactose or 3-OMG. CONCLUSIONS. These data indicate that elevations in ambient glucose result in greater metabolism of glucose through glycolysis and polyol metabolism. Induction of AR2 was greatest when RPE cells were grown in pathophysiological concentrations of glucose. Hyperosmolar stress is not a necessary determinant of AR2 mRNA, AR2 protein, or AR2 protein activity in cells that form the outer blood-retinal barrier. Increased facilitative glucose transport or glucose metabolism appears to be requisite for glucose-specific and nonosmotic regulation of AR2 in the RPE cell in vitro.

Exocytosis in the dissociated pancreatic acinar cells of the guinea pig directly visualized by VEC-DIC microscopy.

To elucidate the detailed process of exocytosis at the highest possible accuracy, we dissociated the pancreatic acinus of the guinea pig and observed zymogen granules under a video-enhanced contrast differential interference contrast (VEC-DIC) microscope. The preparation was thin enough to resolve each zymogen granule with the best clarity. When acinar cells were stimulated with ACh (20 microM), many zymogen granules near the lumen showed an abrupt light intensity change. For a period of 10 s immediately before exocytosis, zymogen granules neither shifted their position nor altered their shape within an accuracy of 38 nm. The time required for individual granules to change the light intensity (the releasing time) ranged from 0.15 to 0.70 s. After each response, the granule maintained its altered contrast for a few seconds until it was retrieved to a planar membrane. No compound exocytosis including granule-granule fusion was observed. We concluded that the exocytosis is not directly initiated by any supramolecular change but by a purely molecular event.

Glucose transporters control gene expression of aldose reductase, PKCalpha, and GLUT1 in mesangial cells in vitro.

The process linking increased glucose utilization and activation of metabolic pathways leading to end-organ damage from diabetes is not known. We have previously described rat mesangial cells that were transduced to constitutively express the facilitative glucose transporter 1 (GLUT1, MCGT1 cells) or bacterial beta-galactosidase (MCLacZ, control cells). Glucose transport was rate limiting for extracellular matrix production in the MCGT1 cells. In the present work, we investigated the effect of GLUT1 overexpression in mesangial cells on aldose reductase (AR), protein kinase Calpha (PKCalpha), and native GLUT1 transcript levels, to determine whether changes in GLUT1 alone could regulate their expression in the absence of high extracellular glucose concentrations. MCGT1 cells grown in normal (8 mM) or elevated (20 mM) glucose had elevated abundance of AR, PKCalpha, and the native GLUT1 transcripts compared with control cells. AR protein levels, AR activity, sorbitol production, and PKCalpha protein content were also greater in the MCGT1 cells than in control cells grown in the same media. This is the first report of the concomitant activation of AR, PKCalpha, and GLUT1 genes by enhanced GLUT1 expression. We conclude that increased GLUT1 expression leads to a positive feedback of greater GLUT1 expression, increased AR expression and activity with polyol accumulation, and increased total and active PKCalpha protein levels, which leads to detrimental stimulation of matrix protein synthesis by diabetic mesangial cells.