Successful islet transplantation depends on the infusion of sufficiently large quantities of islets, of which only ϳ30% become stably engrafted. Rapid and adequate revascularization of transplanted islets is important for islet survival and function. Delayed and insufficient revascularization can deprive islets of oxygen and nutrients, resulting in islet cell death and early graft failure. To improve islet revascularization, we delivered human vascular endothelial growth factor (VEGF) cDNA to murine islets, followed by transplantation under the renal capsule in diabetic mice. Diabetic animals receiving a marginal mass of 300 islets that were pretransduced with a VEGF vector exhibited near normoglycemia. In contrast, diabetic mice receiving an equivalent number of islets that were transduced with a control vector remained hyperglycemic. Immunohistochemistry with anti-insulin and anti-CD31 antibodies revealed a relatively higher insulin content and greater degree of microvasculature in the VEGF vector-transduced islet grafts, which correlated with significantly improved blood glucose profiles and enhanced insulin secretion in response to glucose challenge in this group of diabetic recipient mice. These results demonstrate that VEGF production in islets stimulates graft angiogenesis and enhances islet revascularization. This mechanism might be explored as a novel strategy to accelerate islet revascularization and improve longterm survival of functional islet mass posttransplantation. Diabetes 53: [963][964][965][966][967][968][969][970] 2004

Dietary advanced glycosylation end products (AGEs) have been linked to insulin resistance in db/db(؉؉) mice. To test whether dietary AGEs play a role in the progression of insulin resistance in normal mice fed high-fat diets, normal C57/BL6 mice were randomly assigned to high-fat diets (35% g fat), either high (HAGE-HF group; 995.4 units/mg AGE) or low (by 2.4-fold LAGE-HF group; 329.6 units/mg AGE) in AGE content for 6 months. Age-matched C57/BL6 and db/db (؉؉) mice fed regular diet (5% g fat, 117.4 units/mg AGE) served as controls. After 6 months, 75% of HAGE-HF mice were diabetic and exhibited higher body weight (P < 0.001), fasting glucose (P < 0.001), insulin (P < 0.001), and serum AGEs (P < 0.01) than control mice, while none of the LAGE-HF mice were diabetic despite a similar rise in body weight and plasma lipids. The HAGE-HF group displayed markedly impaired glucose and insulin responses during glucose tolerance tests and euglycemic and hyperglycemic clamps and altered pancreatic islet structure and function compared with those of LAGE-HF mice, in which findings resembled those of control mice. The HAGE-HF group had more visceral fat (by two-and fourfold) and more AGE-modified fat (by two-and fivefold) than LAGE-HF and control mice, respectively. In the HAGE-HF group, plasma 8-isoprostane was higher (P < 0.01) and adiponectin lower (P < 0.001) than control mice, while in the LAGE-HF group, these were more modestly affected (P < 0.05). These results demonstrate that the development of insulin resistance and type 2 diabetes during prolonged high-fat feeding are linked to the excess AGEs/advanced lipoxidation end products inherent in fatty diets. Diabetes 54: 2314 -2319, 2005

Mitochondria-mediated production of reactive oxygen species (ROS) plays a key role in apoptosis. Mitochondrial phospholipid cardiolipin molecules are likely the main target of ROS because they are particularly rich in polyunsaturated fatty acids. They are also located in the inner mitochondrial membrane near the ROS-producing sites. Under physiological conditions mitochondria can repair peroxidative damage in part through a remodeling mechanism via the deacylation-reacylation cycle

Mitochondrial production of reactive oxygen species and oxidation of cardiolipin are key events in initiating apoptosis. We reported that group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) localizes in and protects beta-cell mitochondria from oxidative damage during staurosporine-induced apoptosis. Here, we used iPLA(2)beta-null (iPLA(2)beta(-/-)) mice to investigate the role of iPLA(2)beta in the repair of mitochondrial membranes. We show that islets isolated from iPLA(2)beta(-/-) mice are more sensitive to staurosporine-induced apoptosis than those from wild-type littermates and that 2 wk of daily ip administration of staurosporine to iPLA(2)beta(-/-) mice impairs both the animals' glucose tolerance and glucose-stimulated insulin secretion by their pancreatic islets. Moreover, the iPLA(2)beta inhibitor bromoenol lactone caused mitochondrial membrane peroxidation and cytochrome c release, and these effects were reversed by N-acetyl cysteine. The mitochondrial antioxidant N-t-butyl hydroxylamine blocked staurosporine-induced cytochrome c release and caspase-3 activation in iPLA(2)beta(-/-) islets. Furthermore, the collapse of mitochondrial membrane potential in INS-1 insulinoma cells caused by high glucose and fatty acid levels was attenuated by overexpressing iPLA(2)beta. Interestingly, iPLA(2)beta was expressed only at low levels in islet beta-cells from obesity- and diabetes-prone db/db mice. These findings support the hypothesis that iPLA(2)beta is important in repairing oxidized mitochondrial membrane components (e.g. cardiolipin) and that this prevents cytochrome c release in response to stimuli that otherwise induce apoptosis. The low iPLA(2)beta expression level in db/db mouse beta-cells may render them vulnerable to injury by reactive oxygen species.

Japanese encephalitis (JE) remains the leading cause of viral encephalitis in the Asia-Pacific region, and the live vaccine SA14-14-2 is currently recommended by WHO and widely used in Asian countries with a good safety and efficacy profile. In this study, we demonstrated that SA14-14-2 failed to produce NS1', the larger NS1-related protein, compared with its parental strain SA14 in various cells. Sequence analysis and secondary structure prediction identified a single silent mutation G66A in the NS2A-coding region of SA14-14-2 destabilized the conserved pseudoknot structure, which was associated with a "1 ribosomal frame shift event. Using reverse genetic technology and animal study, we provided solid evidence that this single silent mutation G66A in the NS2A gene abolished the production of NS1' in vitro and reduced neurovirulence and neuroinvasiveness in mice. These findings provide critical information in understanding the molecular mechanism of JE vaccine attenuation and is critical for JE vaccine quality control.