However, recent studies have demonstrated that circulating bone marrow-derived endothelial progenitor cells (EPCs) tightly contribute to adult blood vessel formation (4,5). The EPCs promote in vivo re-endothelization and are able to be incorporated into new vessels in animal models of hind limb ischemia (6,7). EPCs are involved in processes like myocardial ischemia and infarction, wound healing, and endogenous endothelial repair (8 -11). Furthermore, in vivo studies in animal models and in vitro studies using EPCs from type 1 diabetic patients revealed a potential role for glucotoxicity in impairing EPC function (7,(12)(13)(14).High glucose induces pathological alterations through increased formation of advanced glycosylation end product, activation of aldose reductase and protein kinase C, and increased flux through the hexosamine pathway. All of these mechanisms seem to reflect a single hyperglycemiainduced process of overproduction of superoxide anion by the mitochondrial electron transport chain (15). Superoxide inhibits the glycolytic enzyme glyceraldehyde phosphate dehydrogenase, diverting upstream metabolites from glycolysis toward the glucose-driven signaling pathways that cause hyperglycemic damage (16). These processes may be in part reduced by transketolase activation through its cofactor thiamine. In fact, both thiamine and benfotiamine have been shown to correct microvascular and macrovascular complications of diabetes, although at a different extent, blocking three major pathways of hyperglycemic damage (16 -19).Interestingly, the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway is crucial for both endothelial cell function and EPC differentiation (20 -22). The PI 3-kinase/ Akt pathway is known to direct cellular processes like differentiation and stress resistance through a tight regulation of the forkhead family of transcription factors (FoxO1/3a/4). FoxO1 and FoxO3a were recently found to play a role in angiogenesis and vasculogenesis (23-26). We and others have recently shown that both genetic and metabolic factors impair activation of the PI 3-kinase/Akt/ FoxO pathway in mature endothelial cells (27)(28)(29). In this study, we investigated the impact of glucose toxicity on the ability of EPCs to differentiate into mature endothelial cells, and we also tested benfotiamine capacity to bypass the negative effects of high glucose concentrations. RESEARCH DESIGN AND METHODSEPC isolation and culture. EPCs were obtained by isolating peripheral mononuclear cells from human blood buffy coats using Ficoll density centrifugation. Recovered cells were washed twice with PBS. Unselected mononuclear cells were plated on fibronectin-coated culture dishes (Biocoat; Becton Dickinson Labware) at a density of 10 6 cells/ml in Medium 199 (Invitrogen), supplemented with 20% fetal bovine serum, 100 units/ml penicillin/streptomycin (Invitrogen), and 0.05 mg/ml bovine pituitary extract (Invitrogen) and in From the

Tumor necrosis factor ␣-converting enzyme (TACE, also known as ADAM17) was recently involved in the pathogenesis of insulin resistance. We observed that TACE activity was significantly higher in livers of mice fed a high-fat diet (HFD) for 1 month, and this activity was increased in liver > white adipose tissue > muscle after 5 months compared with chow control. In mouse hepatocytes, C 2 C 12 myocytes, and 3T3F442A adipocytes, TACE activity was triggered by palmitic acid, lipolysaccharide, high glucose, and high insulin. TACE overexpression significantly impaired insulin-dependent phosphorylation of AKT, GSK3, and FoxO1 in mouse hepatocytes. To test the role of TACE activation in vivo, we used tissue inhibitor of metalloproteinase 3 (Timp3) null mice, because Timp3 is the specific inhibitor of TACE and Timp3 ؊/؊ mice have higher TACE activity compared with wild-type (WT) mice. Timp3 ؊/؊ mice fed a HFD for 5 months are glucose-intolerant and insulin-resistant; they showed macrovesicular steatosis and ballooning degeneration compared with WT mice, which presented only microvesicular steatosis. Shotgun proteomics analysis revealed that Timp3 ؊/؊ liver showed a significant differential expression of 38 proteins, including lower levels of adenosine kinase, methionine adenosysltransferase I/III, and glycine N-methyltransferase and higher levels of liver fatty acid-binding protein 1. These changes in protein levels were also observed in hepatocytes infected with adenovirus encoding TACE. All these proteins play a role in fatty acid uptake, triglyceride synthesis, and methionine metabolism, providing a molecular explanation for the increased hepatosteatosis observed in Timp3 ؊/؊ compared with WT mice. Conclusion: We have identified novel mechanisms, governed by the TACE-Timp3 interaction, involved in the determination of insulin resistance and liver steatosis during overfeeding in mice. (HEPATOLOGY 2010;51:103-110.) P andemic obesity is now considered the underlying basis for the increasing prevalence of chronic metabolic-inflammatory diseases including type 2 diabetes, nonalcoholic fatty liver disease (NAFLD) and atherosclerosis. 1 Although NAFLD is an emerging metabolic complication of obesity, its pathogenic mechanisms are still unclear. 1 The contribution of insulin resistance to the development of fatty liver occurs in part by deficient control of lipid storage in white adipose tissue and in part by altered

Skeletal integrity is maintained by the co-ordinated activity of osteoblasts, the bone-forming cells, and osteoclasts, the bone-resorbing cells. In this study, we show that mice overexpressing galectin-8, a secreted mammalian lectin of the galectins family, exhibit accelerated osteoclasts activity and bone turnover, which culminates in reduced bone mass, similar to cases of postmenopausal osteoporosis and cancerous osteolysis. This phenotype can be attributed to a direct action of galectin-8 on primary cultures of osteoblasts that secrete the osteoclastogenic factor RANKL upon binding of galectin-8. This results in enhanced differentiation into osteoclasts of the bone marrow cells co-cultured with galectin-8-treated osteoblasts. Secretion of RANKL by galectin-8-treated osteoblasts can be attributed to binding of galectin-8 to receptor complexes that positively (uPAR and MRC2) and negatively (LRP1) regulate galectin-8 function. Our findings identify galectins as new players in osteoclastogenesis and bone remodeling, and highlight a potential regulation of bone mass by animal lectins.DOI: http://dx.doi.org/10.7554/eLife.05914.001

Aberrant activation of Wnt/b-catenin signaling is common in most sporadic and inherited colorectal cancer (CRC) cells leading to elevated b-catenin/TCF transactivation. We previously identified the neural cell adhesion molecule L1 as a target gene of b-catenin/TCF in CRC cells. Forced expression of L1 confers increased cell motility, invasion, and tumorigenesis, and the induction of human CRC cell metastasis to the liver. In human CRC tissue, L1 is exclusively localized at the invasive front of such tumors in a subpopulation of cells displaying nuclear b-catenin. We determined whether L1 expression confers metastatic capacities by inducing an epithelial to mesenchymal transition (EMT) and whether L1 cosegregates with cancer stem cell (CSC) markers. We found that changes in L1 levels do not affect the organization or expression of E-cadherin in cell lines, or in invading CRC tissue cells, and no changes in other epithelial or mesenchymal markers were detected after L1 transfection. The introduction of major EMT regulators (Slug and Twist) into CRC cell lines reduced the levels of E-cadherin and induced fibronectin and vimentin, but unlike L1, Slug and Twist expression was insufficient for conferring metastasis. In CRC cells L1 did not specifically cosegregate with CSC markers including CD133, CD44, and EpCAM. L1-mediated metastasis required NF-kB signaling in cells harboring either high or low levels of endogenous E-cadherin. The results suggest that L1-mediated metastasis of CRC cells does not require changes in EMT and CSC markers and operates by activating NF-kb signaling. Mol Cancer Res; 9(1); 14-24. Ó2010 AACR.