B cell chronic lymphocytic leukemia (B-CLL) consists of the accumulation of malignant cells that apparently escape normal apoptotic regulation. We have studied the role of ␣4␤1 integrin/fibronectin interaction in preventing apoptosis of these cells in vitro. B cells from 16 patients showed constant expression of ␣4␤1 and little or no ␣5␤1. B-CLL cells cultured on fibronectin or two previously described fibronectin recombinant fragments (H89 and H0) which contain the ligands for ␣4␤1, consistently showed higher viability than control cells cultured on poly-lysine. The H89 fragment, containing the high affinity ligand CS-1, was the most efficient substrate with mean cell viability values of 72, 60 and 35% at days 2, 5 and 8 of culture, respectively. For control cells these values were 40, 27 and 15%, respectively. Parallel cell cycle analysis confirmed these results. The anti-apoptotic effect required direct contact with immobilized substrata since it was not observed when using B-CLL conditioned media alone or when clustering ␣4␤1 with specific mAbs in suspension. Quantitation of the apoptosis regulatory proteins Bcl-2 and Bax revealed that cells cultured on the H89 fragment showed high/moderate levels of Bcl-2 (with some interpatient variation) and low levels of Bax resulting in an elevated Bcl-2/Bax ratio. These results indicate that adhesion of B-CLL cells to fibronectin upregulate the Bcl-2/Bax ratio and this may contribute to the anti-apoptotic effect induced via ␣4␤1 integrin.

The effect of magnesium deficiency on glucose disposal, glucose-stimulated insulin secretion and insulin action on skeletal muscle was investigated in rats which were fed a low magnesium-containing diet for 4 days. Control rats were fed a standard diet. Compared to the control rats, the rats fed with low magnesium diet presented: 1) lower serum magnesium levels (0.45 +/- 0.02 vs 0.78 +/- 0.01 mmol/l, p < 0.001), 2) higher basal serum glucose (6.8 +/- 0.02 vs 5.5 +/- 0.2 mmol/l, p < 0.05) and similar basal serum insulin, 3) 40% reduction (p < 0.001) in the glucose disappearance rate after its i.v. administration, and 4) 45% reduction (p < 0.05) in the glucose-stimulated insulin secretion. The insulin action upon the glucose uptake by skeletal muscle was determined by means of hindquarter perfusions. Compared with control rats, magnesium-deficient rats presented: 1) normal basal glucose uptake, 2) lower stimulatory effect on the glucose uptake by insulin at the concentrations of 5 x 10(-10) mol/l (3.0 +/- 0.9 vs 5.4 +/- 0.6, p < 0.05) and 5 x 10(-9) mol/l (6.3 +/- 0.5 vs 8.0 +/- 0.5, p < 0.05), 3) normal glucose uptake at a maximal insulin concentration of 1 x 10(-7) mol/l, and 4) 50% reduction in the insulin sensitivity (ED50: 1.3 +/- 0.3 vs 0.55 +/- 0.1 mol/l, p < 0.05). In partially purified insulin receptors prepared from gastrocnemius muscle, 125I-insulin binding was similar in both groups of rats. However, the autophosphorylation of the beta-subunit of the insulin receptor was significantly reduced by 50% in magnesium-deficient rats and the tyrosine kinase activity of insulin receptors toward the exogenous substrate Poly Glu4; Tyr 1 was also reduced (p < 0.05) by hypomagnesaemia. The abundance of the insulin-sensitive glucose transporter protein (muscle/fat GLUT4), measured by Western blot analysis using polyclonal antisera, was similar in muscles of control and hypomagnesaemic rats. These findings indicate that hypomagnesaemia has a deleterious effect on glucose metabolism due to an impairment of both insulin secretion and action. The insulin resistance observed in skeletal muscle of magnesium-deficient rats may be attributed, at least in part, to a defective tyrosine kinase activity of insulin receptors.

SummaryThe effect of magnesium deficiency on glucose disposal, glucose-stimulated insulin secretion and insulin action on skeletal muscle was investigated in rats which were fed a low magnesium-containing diet for 4 days. Control rats were fed a standard diet. Compared to the control rats, the rats fed with low magnesium diet presented: 1) lower serum magnesium levels (0.45 + 0.02 vs 0.78 + 0.01 mmol/1, p < 0.001), 2) higher basal serum glucose (6.8 +__ 0.2 vs 5.5 + 0.2 mmol/1, p < 0.05) and similar basal serum insulin, 3) 40 % reduction (p < 0.001) in the glucose disappearance rate after its i.v. administration, and 4) 45 % reduction (p < 0.05) in the glucose-stimulated insulin secretion. The insulin action upon the glucose uptake by skeletal muscle was determined by means of hindquarter perfusions. Compared with control rats, magnesium-deficient rats presented: 1) normal basal glucose uptake, 2) lower stimulatory effect on the glucose uptake by insulin at the concentrations of 5 x 10 -l~ mol/1 (3.0 + 0.9 vs 5.4 + 0.6, p < 0.05) and 5 x 10 -9 tool/1 (6.3 + 0.5 vs 8.0 + 0.5, p < 0.05), 3) normal glucose uptake at a maximal insulin concentration of i x 10 .7 mol/1, and 4) 50 % reduction in the insulin sensitivity (ED50:1.3 +_ 0.3 vs 0.55 + 0.1 mol/1, p < 0.05). In partially purified insulin receptors prepared from gastrocnemius muscle, 125I-insulin binding was similar in both groups of rats. However, the autophosphorylation of the fi-subunit of the insulin receptor was significantly reduced by 50 % in magnesium-deficient rats and the tyrosine kinase activity of insulin receptors toward the exogenous substrate Poly Glu4: Tyr 1 was also reduced (p < 0.05) by hypomagnesaemia. The abundance of the insulin-sensitive glucose transporter protein (muscle/fat GLUT4), measured by Western blot analysis using polyclonal antisera, was similar in muscles of control and hypomagnesaemic rats. These findings indicate that hypomagnesaemia has a deleterious effect on glucose metabolism due to an impairment of both insulin secretion and action. The insulin resistance observed in skeletal muscle of magnesium-deficient rats may be attributed, at least in part, to a defective tyrosine kinase activity of insulin receptors.

We have studied the function of the Hep III fibronectin domain in the cytoskeletal response initiated by alpha5beta1 integrin-mediated adhesion. Melanoma cells formed stress fibers and focal adhesions on the RGD-containing FNIII7-10 fragment. Coimmobilization of FNIII4 -5, a fragment spanning Hep III and containing the alpha4beta1 ligand H2 with FNIII7-10, or addition of soluble FNIII4 -5 to cells preattached to FNIII7-10, inhibited stress fibers and induced cytoplasmic protrusions. This effect involved alpha4beta1 since: 1) mutations in H2 reverted the inhibition; 2) other alpha4beta1 ligands (CS-1, VCAM-1), an anti-alpha4 mAb, or alpha4 expression in HeLa cells inhibited stress fibers. This activity was apparently cryptic in fibronectin or large fibronectin fragments, but exposed upon proteolytic degradation. Indeed purified peptic fragments containing H2, inhibited stress fibers when mixed with FNIII7-10 or fibronectin. RhoA activation with LPA or transfection with V14RhoA reverted the inhibitory effect and induced stress fibers on FNIII7-10ϩFNIII4 -5. Furthermore, addition of alpha4beta1 ligands to FNIII7-10, down-regulated RhoA and activated p190RhoGAP, which localized to cytoplasmic protrusions. alpha4beta1/ligand interaction induced cell migration, monitored by video microscopy and wound healing assays. These data indicate that alpha4beta1 provides an antagonistic signal to alpha5beta1 by interfering with the RhoA activation pathway and this leads to melanoma cell migration.