Genetic engineering of non-beta cells to release insulin upon feeding could be a therapeutic modality for patients with diabetes. A tumor-derived K-cell line was induced to produce human insulin by providing the cells with the human insulin gene linked to the 5'-regulatory region of the gene encoding glucose-dependent insulinotropic polypeptide (GIP). Mice expressing this transgene produced human insulin specifically in gut K cells. This insulin protected the mice from developing diabetes and maintained glucose tolerance after destruction of the native insulin-producing beta cells.

OBJECTIVE-Type 1 diabetes is associated with increased microvascular complications and inflammation. The monocytemacrophage is a pivotal cell in atherogenesis. There are scanty data on noninvasive measures of microvascular abnormalities and inflammation in type 1 diabetic subjects with microvascular complications. Thus, we examined systemic and cellular biomarkers of inflammation in type 1 diabetic patients with microvascular complications (T1DM-MV patients) and type 1 diabetic patients without microvascular complications (T1DM patients) compared with matched control subjects and determined the microcirculatory abnormalities in the T1DM and T1DM-MV patients using computer-assisted intravital microscopy (CAIM).RESEARCH DESIGN AND METHODS-Fasting blood, 24-h urine, and CAIM measurements were obtained from the T1DM and T1DM-MV patients and matched control subjects. C-reactive protein, E-selectin, nitrotyrosine, monocyte superoxide, and cytokines were elevated in the T1DM and T1DM-MV patients compared with control subjects (P Ͻ 0.01).RESULTS-Severity index, as assessed by CAIM, was significantly increased in the T1DM and T1DM-MV patients compared with the control subjects (P Ͻ 0.001). There was a significant increase in C-reactive protein, nitrotyrosine, vascular cell adhesion molecule and monocyte superoxide anion release, and interleukin-1 release in T1DM-MV compared with T1DM patients (P Ͻ 0.05). T1DM-MV patients had significantly increased CAIM severity index and microalbumin-to-creatinine ratio compared with T1DM patients (P Ͻ 0.05). Furthermore, pp38MAPK, pp65, and pERK activity were significantly increased in monocytes from the T1DM and T1DM-MV patients compared with those from the controls subjects, and pp38MAPK and pp65 activity were significantly increased in the T1DM-MV compared with the T1DM patients (P Ͻ 0.01). C oronary artery disease is the main cause of death in type 1 diabetes. Type 1 diabetes is associated with an increased risk of vascular complications, and type 1 diabetic patients with proteinuria and/or retinopathy have a significantly increased risk of fatal coronary artery disease (1). Most studies have indicated that this excess risk for macrovascular complications cannot be explained solely by conventional risk factors such as dyslipidemia, hypertension, and smoking. Therefore, the diabetic state per se confers an increased propensity to accelerated atherogenesis. However, the precise mechanisms remain to be elucidated. Inflammation is pivotal in atherosclerosis (2). The monocyte-macrophage, a crucial cell in atherogenesis, is readily accessible for study. We and others have previously shown that monocytes from type 2 diabetic patients with and without complications exhibit increased proatherogenic activity compared with matched control subjects (3)(4)(5). Recently, we demonstrated that type 1 diabetic subjects exhibit increased inflammation as evidenced by increased plasma C-reactive protein (CRP) levels and increased monocyte pro-atherogenic activity (6). However, there are scanty data on...

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Microvascular occlusion in sickle cell disease can be initiated by adhesion of sickle red blood cells (RBCs) to the endothelium. Our objective in this study was to verify the relevance in vivo of our discovery that sickle RBCs adhere abnormally to endothelial P-selectin in vitro. We used computer-assisted intravital microscopy to characterize RBC flow velocity (V RBC ) in mice. We found faster V RBC of sickle RBCs in P-selectin knock-out and control mice than in sickle cell mice, which have increased endothelial cell P-selectin expression. Agonist peptide for murine protease-activated receptor-1 (PAR-1), which selectively activates mouse endothelial cells but not platelets, was used to assess the effects of endothelial cell Pselectin on microvascular flow. Suffusion of venules with this agonist stopped flow promptly in normal and sickle mice but not in P-selectin knock-out mice or in control mice pretreated with anti-P-selectin monoclonal antibody or unfractionated heparin (UFH). Agonist-induced slowing of flow was reversed rapidly by suffusion with UFH, provided flow had not already stopped. We conclude that endothelial cell P-selectin contributes to the microcirculatory abnormalities in sickle cell disease and that blocking Pselectin may be useful for preventing painful vasoocclusion in sickle cell disease. ( IntroductionMost of the morbid consequences of sickle cell disease are caused by the impairment of blood flow in the microvasculature. 1 Traditional understandings attribute the microvascular occlusion to an increase in blood viscosity caused by intraerythrocytic polymerization of deoxygenated sickle hemoglobin and the consequent sickling and rigidification of red blood cells (RBCs). 2 Kinetic considerations predict that, in the absence of preexisting intraerythrocytic polymer, impairment of blood viscosity will occur after the RBC has entered into veins too large to be occluded by rigid sickled RBCs 3 and that polymerization will occur within vessels small enough to be occluded by individual sickled RBCs only when their transit through the microcirculation is delayed.Among the factors that can prolong the transit time of sickle RBCs through the microvasculature are several polymerizationindependent processes, including vascular constriction, coagulation, inflammation, and cellular adhesion. 4 Experimental evidence supports a 2-step mechanism of vasoocclusion initiated by the binding of an adhesive subset of sickle RBCs to the vascular endothelium and completed by the logjamming of more rigid sickle RBCs behind the adherent nidus. 5 This discovery has provided an understanding of the onset of painful vasoocclusion that is lacking from detailed explications of hemoglobin S polymerization and RBC sickling and notions of systemic deoxygenation 6 and inspired a profusion of research into mechanisms of adhesion and adhesion-blocking therapies. 7,8 The expression of cytoadhesion molecules on endothelial cells isolated from the circulating blood of patients with sickle cell disease 9 and on intact endothelial c...

Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce insulin resistance in cultured cells as well as in animal models. The aim of this study was to map the in vivo mechanism whereby TNF-alpha contributes to the pathogenesis of impaired insulin signaling, using obese and lean Zucker rats in which TNF-alpha activity was inhibited through adenovirus-mediated gene transfer. We employed a replication-incompetent adenovirus-5 (Ad5) vector to endogenously express a TNF inhibitor (TNFi) gene, which encodes a chimeric protein consisting of the extracellular domain of the human 55-kDa TNF receptor joined to a mouse IgG heavy chain. Control animals consisted of rats infected with the same titer of adenovirus carrying the lac-z complementary DNA, encoding for beta-galactosidase. There was a significant reduction in plasma insulin and free fatty acid levels in TNFi obese rats 2 days following Ad5 administration. The peripheral insulin sensitivity index was 50% greater, whereas hepatic glucose output was completely suppressed during hyperinsulinemic glucose clamps in TNFi obese animals, with no differences observed between the two lean groups. The improvement in peripheral and hepatic sensitivity to insulin seen in the obese animals was independent of insulin receptor (IR) number and insulin binding affinity for IR. However, TNF-alpha neutralization led to a 2.5-fold increase in tyrosine phosphorylation of IR in skeletal muscle, whereas this was unchanged in liver. There was also a 4-fold increase in particulate protein tyrosine phosphatase activity of skeletal muscle in TNFi obese animals vs. beta-galactosidase controls, whereas protein tyrosine phosphatase activity in liver was unchanged. These results suggest that TNF-alpha is a mediator of insulin resistance in obesity and may modulate IR signaling in skeletal muscle and liver through different pathways. TNF-alpha may affect insulin action in the liver either at sites distal to the IR or indirectly, possibly because of increased provision of gluconeogenic substrates or altered counterregulation. In addition, the Ad5-mediated gene delivery system employed here provides an in vivo model that is efficient and economical for exploring mechanisms involved in TNF-alpha-induced insulin resistance in various genetic models of obesity-linked diabetes.