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BACKGROUND:A growing body of literature on Roux-en-Y gastric bypass surgery (RYGB) has generated inconclusive results on the mechanism underlying the beneficial effects on weight loss and glycaemia, partially due to the problems of designing clinical studies with the appropriate controls. Moreover, RYGB is only performed in obese individuals, in whom metabolism is perturbed and not completely understood. METHODS:In an attempt to isolate the effects of RYGB and its effects on normal metabolism, we investigated the effect of RYGB in lean pigs, using sham-operated pair-fed pigs as controls. Two weeks post-surgery, pigs were subjected to an intravenous glucose tolerance test (IVGTT) and circulating metabolites, hormones and lipids measured. Bile acid composition was profiled after extraction from blood, faeces and the gallbladder. RESULTS:A similar weight development in both groups of pigs validated our experimental model. Despite similar changes in fasting insulin, RYGB-pigs had lower fasting glucose levels. During an IVGTT RYGB-pigs had higher insulin and lower glucose levels. VLDL and IDL were lower in RYGB- than in sham-pigs. RYGB-pigs had increased levels of most amino acids, including branched-chain amino acids, but these were more efficiently suppressed by glucose. Levels of bile acids in the gallbladder were higher, whereas plasma and faecal bile acid levels were lower in RYGB- than in sham-pigs. CONCLUSION:In a lean model RYGB caused lower plasma lipid and bile acid levels, which were compensated for by increased plasma amino acids, suggesting a switch from lipid to protein metabolism during fasting in the immediate postoperative period.

Type 2 diabetic patients suffer from insulin resistance and reduced insulin secretion. Osteopontin (OPN), a versatile protein expressed in several tissues throughout the body including the islets of Langerhans, has previously been implicated in the development of insulin resistance. Here we have investigated the role of OPN in insulin secretion using an OPN knock out mouse model (OPN-/-). Ultra-structural analyzes of islets from OPN-/- and WT mice indicated weaker cell-cell connections between the islet cells in the OPN-/- mouse compared to WT. Analysis of the insulin granule distribution in the beta cells showed that although OPN-/- and WT beta cells have the same number of insulin granules OPN-/- beta cells have significantly fewer docked granules. Both OPN-/- and WT islets displayed synchronized Ca2+ oscillations indicative of an intact beta cell communication. OPN-/- islets displayed higher intracellular Ca2+ concentrations when stimulated with 16.7 mM glucose than WT islets and the initial dip upon elevated glucose concentrations (which is associated with Ca2+ uptake into ER) was significantly lower in these islets. Glucose-induced insulin secretion was similar in OPN-/- and WT islets. Likewise, non-fasted blood glucose levels were the same in both groups. In summary, deletion of OPN results in several minor beta-cell defects that can be compensated for in a healthy system.

OBJECTIVE OF THE STUDY: Diabetic patients have a much more widespread and aggressive form of atherosclerosis and therefore, higher risk for myocardial infarction, peripheral vascular disease and stroke, but the molecular mechanisms leading to accelerated damage are still unclear. Recently, we showed that hyperglycemia activates the transcription factor NFAT in the arterial wall, inducing the expression of the pro-atherosclerotic protein osteopontin. Here we investigate whether NFAT activation may be a link between diabetes and atherogenesis. METHODOLOGY AND PRINCIPAL FINDINGS: Streptozotocin (STZ)-induced diabetes in apolipoprotein E(-/-) mice resulted in 2.2 fold increased aortic atherosclerosis and enhanced pro-inflammatory burden, as evidenced by elevated blood monocytes, endothelial activation- and inflammatory markers in aorta, and pro-inflammatory cytokines in plasma. In vivo treatment with the NFAT blocker A-285222 for 4 weeks completely inhibited the diabetes-induced aggravation of atherosclerosis, having no effect in non-diabetic mice. STZ-treated mice exhibited hyperglycemia and higher plasma cholesterol and triglycerides, but these were unaffected by A-285222. NFAT-dependent transcriptional activity was examined in aorta, spleen, thymus, brain, heart, liver and kidney, but only augmented in the aorta of diabetic mice. A-285222 completely blocked this diabetes-driven NFAT activation, but had no impact on the other organs or on splenocyte proliferation or cytokine secretion, ruling out systemic immunosuppression as the mechanism behind reduced atherosclerosis. Instead, NFAT inhibition effectively reduced IL-6, osteopontin, monocyte chemotactic protein 1, intercellular adhesion molecule 1, CD68 and tissue factor expression in the arterial wall and lowered plasma IL-6 in diabetic mice. CONCLUSIONS: Targeting NFAT signaling may be a novel and attractive approach for the treatment of diabetic macrovascular complications.

AIMS: Sight-threatening diabetic retinopathy has been treated with photocoagulation for decades but the mechanisms behind the beneficial clinical effects are poorly understood. One target of irradiation and a potential player in this process is the retinal pigment epithelium (RPE). Here we establish an in vitro model for photocoagulation of human RPE cells. METHODS: ARPE-19 cells were exposed to photocoagulation and studied at various time points up to 168h. Lesion morphology, necrosis and apoptosis were investigated by light microscopy; LIVE/DEAD staining and measurements of lactate dehydrogenase activity; and TUNEL- and ELISA-based quantification of DNA fragments, respectively. Cell migration and proliferation were explored using docetaxel and mitomycin C; temporal and spatial changes in proliferation were assessed by confocal immunofluorescence of proliferating cell nuclear antigen. Gene expression was measured by qPCR. RESULTS: Photocoagulation of ARPE-19 resulted in denaturation of proteins and reproducible lesion formation. A transient peak in necrosis, followed by a peak in apoptosis was observed in cells within the lesions at 6h and 24h, respectively after photocoagulation. Cell proliferation was depressed during the first hours after photocoagulation, back to control levels at 24h and augmented in the following days. These effects were not limited to cells in the lesions, but also evident in neighbouring cells. Changes in cell proliferation during lesion repair were preceded by changes in cell migration. Altered mRNA expression of genes previously implicated in the regulation of cell proliferation (FOS, IL-1β, IL-8, HMGA2), migration and tissue repairing (TGFBR2, ADAMTS6, TIMP3, CTGF) was observed, as well as increased expression of the alarmin IL33 and the cytoprotective gene HSPA6. CONCLUSIONS: Using a laser system and experimental settings that comply with standards used in clinical practice, we have established a suitable model for in vitro photocoagulation of human RPE cells to isolate their contribution to the beneficial effects of laser treatment.

AIM: T cells have been attributed an important role in modulating repair responses following vascular injury. The aim of this study was to investigate the role of different T cell subsets in this context. METHODS AND RESULTS: A non-obstructive collar was introduced to inflict carotid artery injury in mice and subsequent activation of immune cells in draining lymph nodes and spleen were studied by flow cytometry. Carotid artery injury of wild type mice was associated with mobilization of both Th1 type CD4(+)IFNγ(+) and regulatory CD4(+)CD25(+)FoxP3(+) T cells in draining lymph nodes. Studies using FoxP3-green fluorescent protein (GFP) transgenic C57/Bl6 mice demonstrated scattered presence of regulatory T cells in the adventitial tissue of injured arteries as well as a massive emigration of regulatory T cells from the spleen in response to carotid injury. However, deletion of antigen presentation to CD4+ T cells (H2(0) mice), as well as deletion of regulatory T cells (through treatment with blocking anti-CD25 antibodies), did not affect neointima formation. Also deletion of antigen presentation to CD8(+) T cells (Tap1(0) mice) was without effect on carotid collar-induced neointima formation. CONCLUSION: The results demonstrate that carotid artery injury is associated with mobilization of regulatory T cells. Depletion of regulatory T cells does not, however, influence the subsequent repair processes leading to the formation of a neointima. The results also demonstrate that lack of CD8(+) T cells does not influence neointima formation in presence of functional CD4(+) T cells and B cells.

BACKGROUND: Inflammation has been proposed to be important in the pathogenesis of diabetic retinopathy. An early feature of inflammation is the release of cytokines leading to increased expression of endothelial activation markers such as vascular cellular adhesion molecule-1 (VCAM-1). Here we investigated the impact of diabetes and dyslipidemia on VCAM-1 expression in mouse retinal vessels, as well as the potential role of tumor necrosis factor-α (TNFα). METHODOLOGY/PRINCIPAL FINDINGS: Expression of VCAM-1 was examined by confocal immunofluorescence microscopy in vessels of wild type (wt), hyperlipidemic (ApoE(-/-)) and TNFα deficient (TNFα(-/-), ApoE(-/-)/TNFα(-/-)) mice. Eight weeks of streptozotocin-induced diabetes resulted in increased VCAM-1 in wt mice, predominantly in small vessels (