Paul N. Epstein

Overexpression of catalase provides partial protection to transgenic mouse beta cells.

Pancreatic beta cells are sensitive to reactive oxygen species and this may play an important role in type 1 diabetes and during transplantation. Beta cells contain low levels of enzyme systems that protect against reactive oxygen species. The weakest link in their protection system is a deficiency in the ability to detoxify hydrogen peroxide by the enzymes glutathione peroxidase and catalase. We hypothesize that the deficit in the ability to dispose of reactive oxygen species is responsible for the unusual sensitivity of beta cells and that increasing protection will result in more resistant beta cells. To test these hypotheses we have produced transgenic mice with increased beta cell levels of catalase. Seven lines of catalase transgenic mice were produced using the insulin promoter to direct pancreatic beta cell specific expression. Catalase activity in islets from these mice was increased by as much as 50-fold. Northern blot analysis of several tissues indicated that overexpression was specific to the pancreatic islet. Catalase overexpression had no detrimental effects on islet function. To test whether increased catalase activity could protect the transgenic islets we exposed them to hydrogen peroxide, streptozocin, and interleukin-1beta. Fifty-fold overexpression of catalase produced marked protection of islet insulin secretion against hydrogen peroxide and significantly reduced the diabetogenic effect of streptozocin in vivo. However, catalase overexpression did not provide protection against interleukin-1beta toxicity and did not alter the effects of syngeneic and allogenic transplantation on islet insulin content. Our results indicate that in the pancreatic beta cell overexpression of catalase is protective against some beta cell toxins and is compatible with normal function.

Causes and Characteristics of Diabetic Cardiomyopathy

Type 1 and type 2 diabetic patients are at increased risk of cardiomyopathy and heart failure is a major cause of death for these patients. Cardiomyopathy in diabetes is associated with a cluster of features including decreased diastolic compliance, interstitial fibrosis and myocyte hypertrophy. The mechanisms leading to diabetic cardiomyopathy remain uncertain. Diabetes is associated with most known risk factors for cardiac failure seen in the overall population, including obesity, dyslipidemia, thrombosis, infarction, hypertension, activation of multiple hormone and cytokine systems, autonomic neuropathy, endothelial dysfunction and coronary artery disease. In light of these common contributing pathologies it remains uncertain whether diabetic cardiomyopathy is a distinct disease. It is also uncertain which factors are most important to the overall incidence of heart failure in diabetic patients. This review focuses on factors that can have direct effects on diabetic cardiomyocytes: hyperglycemia, altered fuel use, and changes in the activity of insulin and angiotensin. Particular attention is given to the changes these factors can have on cardiac mitochondria and the role of reactive oxygen species in mediating injury to cardiomyocytes.

Ultrastructural and functional analyses of nephropathy in calmodulin‐induced diabetic transgenic mice

Previous animal models of diabetic nephropathy have used diabetic animals for which the underlying defect was either uncertain or the diabetes was induced by potentially specific toxins. In this report, we describe the renal abnormalities in a transgenic mouse model that develops early‐onset diabetes due to overexpression of calmodulin in pancreatic beta cells.

Elevated hexokinase increases cardiac glycolysis in transgenic mice

OBJECTIVE Cardiac glucose metabolism is critical to normal and pathological function. The significance of the first committed metabolic step, glucose phosphorylation, has not been established. In this study a new transgenic model was developed in order to investigate the importance of this enzymatic step in cardiac glycolysis. METHODS Transgenic mice were produced that overexpress yeast hexokinase B under the control of a cardiac specific promoter. Yeast hexokinase B is a high affinity enzyme that is not inhibited by glucose-6-phosphate. Hexokinase enzyme activity was measured by a modified radiometric procedure. Cardiac glucose metabolism and contractility were measured in the Langendorff mode. Cardiac glycogen content and glucose-6-phosphate independent glycogen synthase activity were also determined. RESULTS In transgenic hearts hexokinase activity was significantly elevated and increased glucose metabolism, particularly in the presence of insulin and during cardiac reperfusion. However during ischemic perfusion the effect of the transgene on glycolysis was minimal. Under all conditions tested there was no effect of hexokinase on contractility. Glycogen content of transgenic hearts was elevated 2-fold and glucose-6-phosphate independent glycogen synthase was also increased. CONCLUSION These results demonstrate that glucose phosphorylation is a key step in determining cardiac glucose metabolism under oxidative conditions.

Prenatal ethanol exposure alters ventricular myocyte contractile function in the offspring of rats

Fetal alcohol syndrome (FAS) is often associated with cardiac hypertrophy and impaired ventricular function in a manner similar to postnatal chronic alcohol ingestion. Chronic alcoholism has been shown to lead to hypomagnesemia, and dietary Mg2+ supplementation was shown to ameliorate ethanol-induced cardiovascular dysfunction such as hypertension. However, the role of gestational Mg2+ supplementation on FAS-related cardiac dysfunction is unknown. This study was conducted to examine the influence of gestational dietary Mg2+ supplementation on prenatal ethanol exposure-induced cardiac contractile response at the ventricular myocyte level. Timed-pregnancy female rats were fed from gestation day 2 with liquid diets containing 0.13 g/L Mg2+ supplemented with ethanol (36%) or additional Mg2+ (0.52 g/L), or both. The pups were maintained on standard rat chow through adulthood, and ventricular myocytes were isolated and stimulated to contract at 0.5 Hz. Mechanical properties were evaluated using an IonOptix™ soft-edge system, and intracellular Ca2+ transients were measured as changes in fura-2 fluorescence intensity (ΔFFI). Offspring from all groups displayed similar growth curves. Myocytes from the ethanol group exhibited reduced cell length, enhanced peak shortening (PS), and shortened time to 90% relengthening (TR90) associated with a normal ΔFFI and time to PS (TPS). Mg2+ reverted the prenatal ethanol-induced alteration in PS and maximal velocity of relengthening. However, it shortened TPS and TR90, and altered the ΔFFI, as well as Ca2+ decay rate by itself. Additionally, myocytes from the ethanol group exhibited impaired responsiveness to increased extracellular Ca2+ or stimulating frequency, which were restored by gestational Mg2+ supplementation. These data suggest that although gestational Mg2+ supplementation may be beneficial to certain cardiac contractile dysfunctions in offspring of alcoholic mothers, caution must be taken, as Mg2+ supplementation affects cell mechanics itself.

Catalase transaction decreases hydrogen peroxide toxicity in a pancreatic beta cell line

BetaTC6-F7 cells like normal Beta cells were found to be highly sensitive to hydrogen peroxide and to possess very low levels of catalase. Therefore we tested whether overexpression of catalase could enhance resistance to hydrogen peroxide. Enzyme activity was increased forty fold by transient transfection of a catalase transgene. To assess protection from hydrogen peroxide a cotransfection method using a human growth hormone reporter gene was developed. Human growth hormone secretion was shown to be a suitable marker for insulin secretion since both hormones demonstrated virtually identical glucose dose response curves. Catalase transfection was found to provide significant protection against hydrogen peroxide indicating that low catalase may contribute to the sensitivity of cells to hydrogen peroxide.

UNEQUAL POTENCY OF TRANSGENIC YEAST HEXOKINASE ON PANCREATIC BETA CELL METABOLISM AND SECRETION

Recent reports have proposed that sequence specific interactions between glucokinase and other beta cell proteins are important to glucokinase regulation of beta cell activity. We have previously reported enhancement of beta cell function by a transgenic hexokinase derived from yeast which has only 30% amino acid sequence homology to glucokinase. To test the functional significance of the amino acid sequence of islet glucokinase we have made a quantitative study of the effect of yeast hexokinase on beta cell glucose metabolism and insulin secretion. Transgenic and normal islets were assayed for hexokinase activity, glucose usage and insulin secretion. Most parameters were measured at six glucose concentrations between 0.5 and 20 mM glucose. Transgenic islet hexokinase activity measured in islet extracts exceeded normal islet hexokinase activity by 31 to 77 percent at different glucose concentrations. At all glucose concentrations tested the percentage increase in transgenic glucose metabolism greatly exceeded the percentage increase in transgenic hexokinase activity. The increase in transgenic glucose metabolism produced a proportional reduction in the threshold for glucose stimulated insulin secretion. However, yeast hexokinase had little if any effect on the first phase of insulin secretion. The finding that metabolism was very sensitive to yeast hexokinase but first phase secretion was not, supports recent proposals that hexokinase and glucokinase may be physically and functionally separated in the beta cell.