Chromium propionate increases insulin sensitivity in horses following oral and intravenous carbohydrate administration.

Forty-eight Quarter Horse geldings (3 to 8 yr of age) were used to determine the effects of dietary chromium (Cr), in the form of Cr propionate (Cr Prop) on insulin sensitivity. Horses were blocked by age, body condition score, and glucose response to concentrate feeding on day 0 and randomly assigned to treatments. Treatments consisted of 0, 2, 4, or 8 mg Cr/d from Cr Prop. Horses were fed daily a concentrate mix at a rate of 0.2 kg/100 kg body weight (BW) and grass hay at 1.75 to 2.0 kg/100 kg BW. All horses were fed the control diet for 7 d prior to the initiation of the study. After an overnight fast, blood samples from the jugular vein were obtained at 0, 2, and 4 h after concentrate feeding on days 0 and 28 for the determination of glucose, nonesterified fatty acids, and insulin. A glucose tolerance test (GTT) was conducted on day 42. Glucose was infused via jugular vein catheters, and blood samples were collected at various times relative to dosing for glucose and insulin determination. Plasma glucose on day 28 was affected (P < 0.05) by treatment, time, and treatment × time. Horses fed 4 mg Cr/d had lesser (P < 0.05) plasma glucose concentrations than those in the other treatments at 0 h. At 2 h post-feeding glucose concentrations were greater (P < 0.05) in horses fed 0 or 8 mg Cr/d than in those given 4 mg Cr. Horses fed 2 mg Cr/d had lesser (P < 0.05) plasma glucose at 4 h post feeding compared with those fed 0 or 8 mg Cr. Plasma glucose did not differ among horses receiving 2 or 4 mg Cr/d at 2 or 4 h. Serum insulin was affected (P < 0.05) by treatment, time, and treatment × time. Insulin concentrations were greater (P < 0.05) in horses fed 0 or 2 mg Cr/d than in those given 4 or 8 mg Cr at 0 h. At 4 h post-feeding insulin concentrations were greater (P < 0.05) in horses given 0 or 8 mg Cr than in those fed 2 or 4 mg Cr/d. Plasma glucose was affected (P < 0.05) by treatment and time, but not by treatment × time following the GTT. Mean plasma glucose (across sampling times) concentrations were greater (P < 0.05) in controls than in horses fed 2 or 4 mg Cr/d. Glucose concentrations following the GTT did not differ among controls and horses given 8 mg Cr/d. Following glucose infusion, serum insulin concentrations were greater (P < 0.05) in horses fed 2 or 4 mg Cr and tended to be greater in those fed 8 mg Cr/d compared with controls. The results of this study indicate that 2 or 4 mg Cr/d from Cr Prop increased insulin sensitivity in adult horses following oral carbohydrate consumption.

Preventing the activation or cycling of the Rap1 GTPase alters adhesion and cytoskeletal dynamics and blocks metastatic melanoma cell extravasation into the lungs.

The Rap1 GTPase is a master regulator of cell adhesion, polarity, and migration. We show that both blocking Rap1 activation and expressing a constitutively active form of Rap1 reduced the ability of B16F1 melanoma cells to extravasate from the microvasculature and form metastatic lesions in the lungs. This correlated with a decreased ability of the tumor cells to undergo transendothelial migration (TEM) in vitro and form dynamic, F-actin-rich pseudopodia that penetrate capillary endothelial walls in vivo. Using multiple tumor cell lines, we show that the inability to form these membrane protrusions, which likely promote TEM and extravasation, can be explained by altered adhesion dynamics and impaired cell polarization that result when Rap1 activation or cycling is perturbed. Thus, targeting Rap1 could be a useful approach for reducing the metastatic dissemination of tumor cells that undergo active TEM.

The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes.

Integrin-mediated adhesion plays an important role in B cell development and activation. Signaling initiated by antigens, chemokines, or phorbol esters can rapidly convert integrins to an activated adhesion-competent state. The binding of integrins to their ligands can then induce actin-dependent cell spreading, which can facilitate cell-cell adhesion or cell migration on extracellular matrices. The signaling pathways involved in integrin activation and post-adhesion events in B cells are not completely understood. We have previously shown that anti-Ig antibodies, the chemokine stromal cell-derived factor-1 (SDF-1; CXCL12), and phorbol esters activate the Rap1 and Rap2 GTPases in B cells and that Rap activation is essential for SDF-1-induced B cell migration (McLeod, S. J., Li, A. H. Y., Lee, R. L., Burgess, A. E., and Gold, M. R. (2002) J. Immunol. 169, 1365-1371; Christian, S. L., Lee, R. L., McLeod, S. J., Burgess, A. E., Li, A. H. Y., Dang-Lawson, M., Lin, K. B. L., and Gold, M. R. (2003) J. Biol. Chem. 278, 41756-41767). We show here that preventing Rap activation by expressing Rap-specific GTPase-activating protein II (RapGAPII) significantly decreased lymphocyte function-associated antigen-1- and alpha(4) integrin-dependent binding of murine B cell lines to purified adhesion molecules and to other cells. Conversely, augmenting Rap activation by expressing a constitutively active form of Rap2 enhanced B cell adhesion, showing for the first time that Rap2 can promote integrin activation. We also show that blocking Rap activation inhibited anti-Ig-induced cell spreading and phorbol ester-induced actin polymerization as well as anti-Ig- and SDF-1-induced phosphorylation of Pyk2, a tyrosine kinase involved in morphological changes and chemokine-induced B cell migration. Thus, the Rap GTPases regulate integrin-mediated B cell adhesion as well as processes that control B cell morphology and migration.

Activation of the Rap GTPases in B lymphocytes modulates B cell antigen receptor-induced activation of Akt but has no effect on MAPK activation.

Signaling by the B cell antigen receptor (BCR) activates the Rap1 and Rap2 GTPases, putative antagonists of Ras-mediated signaling. Because Ras can activate the Raf-1/ERK pathway and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, we asked whether Rap activation limits the ability of the BCR to signal via these pathways. To do this, we blocked the activation of endogenous Rap1 and Rap2 by expressing the Rap-specific GTPase-activating protein RapGAPII. Preventing Rap activation had no effect on BCR-induced activation of ERK. In contrast, BCR-induced phosphorylation of Akt on critical activating sites was increased 2- to 3-fold when Rap activation was blocked. Preventing Rap activation also increased the ability of the BCR to stimulate Akt-dependent phosphorylation of the FKHR transcription factor on negative regulatory sites and decreased the levels of p27Kip1, a pro-apoptotic factor whose transcription is enhanced by FKHR. Moreover, preventing Rap activation reduced BCR-induced cell death in the WEHI-231 B cell line. Thus activation of endogenous Rap by the BCR limits BCR-induced activation of the PI3K/Akt pathway, opposes the subsequent inhibition of the FKHR/p27Kip1 pro-apoptotic module, and enhances BCR-induced cell death. Consistent with the idea that Rap-GTP is a negative regulator of the PI3K/Akt pathway, expressing constitutively active Rap2 (Rap2V12) reduced BCR-induced phosphorylation of Akt and FKHR. Finally, our finding that Rap2V12 can bind PI3K and inhibit its activity in a manner that depends upon BCR engagement provides a potential mechanism by which Rap-GTP limits activation of the PI3K/Akt pathway, a central regulator of B cell growth and survival.

The Rap GTPases regulate B cell migration toward the chemokine stromal cell-derived factor-1 (CXCL12): potential role for Rap2 in promoting B cell migration.

Stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for B cells and B cell progenitors. Although the binding of SDF-1 to its receptor, CXCR4, activates multiple signaling pathways, the mechanism by which SDF-1 regulates cell migration is not completely understood. In this report we show that activation of the Rap GTPases is important for B cells to migrate toward SDF-1. We found that treating B cells with SDF-1 resulted in the rapid activation of both Rap1 and Rap2. Moreover, blocking the activation of Rap1 and Rap2 via the expression of a Rap-specific GTPase-activating protein significantly reduced the ability of B cells to migrate toward SDF-1. Conversely, expressing a constitutively active form of Rap2 increased SDF-1-induced B cell migration. Thus, the Rap GTPases control cellular processes that are important for B cells to migrate toward SDF-1.