Chiarella Bozzo

Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival

Adhesion of human primary skin fibroblasts and ECV304 endothelial cells to immobilized matrix proteins, β1 or αv integrin antibodies stimulates tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. This tyrosine phosphorylation is transiently induced, reaching maximal levels 30 min after adhesion, and it occurs in the absence of receptor ligands. Similar results were observed with EGF receptor‐transfected NIH‐3T3 cells. Use of a kinase‐negative EGF receptor mutant demonstrates that the integrin‐stimulated tyrosine phosphorylation is due to activation of the receptors intrinsic kinase activity. Integrin‐mediated EGF receptor activation leads to Erk‐1/MAP kinase induction, as shown by treatment with the specific inhibitor tyrphostin AG1478 and by expression of a dominant‐negative EGF receptor mutant. EGF receptor and Erk‐1/MAP kinase activation by integrins does not lead per se to cell proliferation, but is important for entry into S phase in response to EGF or serum. EGF receptor activation is also required for extracellular matrix‐mediated cell survival. Adhesion‐dependent MAP kinase activation and survival are regulated through EGF receptor activation in cells expressing this molecule above a threshold level (5×103 receptors per cell). These results demonstrate that integrin‐dependent EGF receptor activation is a novel signaling mechanism involved in cell survival and proliferation in response to extracellular matrix.

Integrin-Mediated Signal Transduction in Human Endothelial Cells: Analysis of Tyrosine Phosphorylation Events

Adhesion of human umbilical endothelial cells to fibronectin resulted in increased tyrosine phosphorylation of a group of proteins with molecular mass ranging from 100 to 130 kDa and of a 70 kDa protein. This pattern of tyrosine phosphorylation was also observed when endothelial cells adhered to vitronectin, collagen IV, collagen I and laminin or to culture dishes coated with antibodies directed to either beta 1, alpha 3, alpha 5, alpha 6 or beta 3 integrin subunits. Increased phosphorylation of the 100-130 kDa proteins was detectable as early as 30 sec after adhesion, reached maximal level after 15 min, and remained high as long as the cells adhere to culture dishes. The 70 kDa protein was phosphorylated with a slower kinetics and its phosphorylation increased over a period of 3 h. Using specific monoclonal antibodies, the major component of the 100-130 kDa complex was identified as the focal adhesion tyrosine kinase p125FAK. The phosphorylation of the p125FAK was also observed by inducing beta 1 integrin clustering in non adherent HEC, indicating that this is a primary signalling event induced by integrins. Using tyrosine kinase inhibitors, we show a direct correlation between integrin-stimulated tyrosine kinases and assembly of focal adhesions and actin fibres.

Insulin resistance in the aged: The role of the peripheral insulin receptors

In the healthy subject, glucose tolerance tends to decrease with age due to impaired insulin secretion and/or decreased peripheral insulin activity. An oral glucose (100 g) tolerance test was performed on 12 aged (70 +/- 4 yr) and 8 young (32 +/- 7 yr) subjects; these subjects underwent laparatomy for cholecystectomy or the management of abdominal diseases. Subcutaneous adipose tissue was removed during surgery and fat cells, prepared according to a personal modification of Rodbells method, were incubated in a medium containing monoiodo- and cold insulin to evaluate insulin binding and affinity constants. The results of the tolerance test pointed to an insulin resistant state i.e., impaired glucose tolerance coupled with normal plasma insulin, as previously shown also by us using other methods in the aged subject. The binding study demonstrates a distinct insulin receptor decrease in fat cells from the older subjects (185,000 +/- 19,200 as opposed to 310,000 +/- 12,000), without any change in affinity constants. The result indicates that insulin resistance in the aged may be attributed at least in part to a reduction in the number of insulin receptors on the target cells. This could be a consequence of aging itself, as proposed by other workers in the case of old fat rats.

Lymphocyte Na,K-ATPase is reduced in aged people

Na,K-ATPase-dependent 86Rb uptake, maximum velocity (Vmax), Michaelis constant (Km) of the uptake, and [3H]-ouabain binding were investigated in the lymphocytes of 10 elderly subjects (age greater than 60 years), and in 10 middle-aged (41 to 60 years) and 10 young controls (age less than or equal to 40 years). 86Rb uptake was reduced in elderly versus both middle-aged and young subjects (20.14 +/- 3.30 v 35.60 +/- 2.67, P = .002, and v 36.53 +/- 4.49 nmol, P = .012), as was the number of [3H]-ouabain binding sites per cell (32,662 +/- 2,215 v 40,420 +/- 1,184, P = .011, and v 40,596 +/- 1,349, P = .014). Vmax was reduced in elderly v young subjects (1.20 +/- 0.10 v 1.64 +/- 0.13, P = .034), but not versus the middle-age group (1.20 +/- 0.10 v 1.54 +/- 0.12 nmol.min-1, NS). Km was no different among the three groups. No differences were found between middle-aged and young subjects. Significant correlations were observed between age and Na,K-ATPase-dependent 86Rb uptake (r = -.620, P = .00009), Vmax (r = -.439, P = .024), and [3H]-ouabain binding sites (r = -.648, P = .002). Moreover, the site number was positively correlated with both uptake (r = .635, P = .002) and Vmax (r = .554, P = .011). These differences were observed both in women and men. We conclude that there is an age-dependent reduction in lymphocyte Na,K-ATPase activity, which is fully manifested over 60 years, and that this alteration is probably due to the reduced number of functional units of Na,K-ATPase in advancing age.

Modulation of extracellular matrix receptors (integrins) on human endothelial cells by cytokines

Integrins are a family of receptor proteins that mediate adhesive interactions [1–3]. Human umbilical vein endothelial cells (HEC) express five distinct integrin complexes that mediate their interaction with basal membrane components [4–7]. We found that tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma), two cytokines known to trigger the inflammatory response and to modify the HEC properties [8], induce selective alterations in the integrin expression. While TNFalpha induces the expression of a laminin-collagen receptor (integrin alpha-1/beta-1), not expressed in unstimulated HEC, a combination of TNFalpha and IFNgamma downregulates the vitronectin receptor (integrin alpha-v/beta-3). These modifications require a minimum of 24 h of exposure to cytokines and reach maximal levels between 48 and 72 h. Such alterations change the adhesive properties of the cells in a selective manner since the expression of other integrins (alpha-3/beta-1 and alpha-5/beta-1) are not altered, and are likely to play a role in the late phase of the inflammatory response.

Dietary guar gum supplementation does not modify insulin resistance in gross obesity

SummaryObesity is considered an insulin resistant state. Dietary guar gum supplementation is able to reduce blood glucose and plasma insulin response to a carbohydrate meal. In order to evaluate whether guar is able to reduce hyperinsulinemia and insulin resistance in gross obesity, we studied 9 obese patients, >50% overweight with impaired glucose tolerance before and after 4+4 g/day guar for 6 weeks. Six patients repeated the treatment with 8+8 g/day guar after a 3-month interval. Guar was added to the usual diet in order to maintain the body weight constant. Pre-treatment and post treatment study included: total specific insulin binding on circulating monocytes; 3H-glucose infusion and euglycemic hyperinsulinemic clamp at ∼100 µU/ml. The differences between post-treatment and pre-treatment values were not significant for any of the parameters studied. Fasting glucose production was: 2.17±0.33 SEM (pretreatment)vs 2.18±0.18 (4+4 g/day)vs 2.28±0.14 (8+8 g/day) mg/kg/min; glucose utilization was: 3.52±0.43vs 3.22±0.44vs 3.49±0.63 mg/kg/min; total specific insulin binding was: 2.80±0.20vs 2.75±0.25vs 2.78±0.31%; body weight was: 101.4±5.4vs 100.2±6.2vs 100.5±7.0 kg. These results indicate that dietary guar gum supplementationper se is unable to reduce insulin resistance in gross obesity if overweight is maintained constant.

Insulin supplement in type 2 diabetic patients with secondary failure to oral agents ameliorates hepatic and peripheral insulin sensitivity but not insulin secretion

In order to investigate the mechanism of amelioration of metabolic abnormalities with supplementary doses of insulin, islet B‐cell function and insulin sensitivity were measured in 10 patients with Type 2 diabetes in secondary failure to oral agents. A small dose of ultralente insulin (0.26 ± 0.07 U kg‐ideal‐body‐weight−1) was added in the morning before breakfast. After 3 months insulin therapy and progressive improvement of metabolic control (HbA1 from 10.5 ± 0.4 to 9.0 ± 0.3 % at the end of insulin treatment, p < 0.001), basal C‐peptide and incremental area during an oral glucose tolerance test were unchanged. In vivo peripheral insulin sensitivity (euglycaemic clamp with insulin infusion of 40, 160, and 600 mU m−2 min−1, respectively) was significantly improved (glucose requirement: to 4.7 ± 1.0 from 3.0 ± 0.6 mg kg−1 min−1, p< 0.05 at first insulin level; to 10.8 ± 0.5 from 9.3 ± 0.7 mg kg−1 min−1, p<0.01 at second level; to 13.3 ± 0.6 from 11.8 ± 0.8 mg kg−1 min−1, p< 0.025 at third level). Basal hepatic glucose production was also significantly reduced (from 4.3 ± 0.4 to 3.3 ± 0.3 mg kg−1 min−1, p< 0.05), and residual glucose production further suppressed after insulin supplement (from 1.1 ± 0.4 to 0.3 ± 0.2 mg kg−1 min−1 after 120 min at 100 mU I−1 plasma insulin, p< 0.05). Specific insulin binding to mononuclear leucocytes was unchanged (from 3.1 ± 0.3 to 3.5 ± 0.3 %, NS).

Insulin secretion and insulin sensitivity defects are a common feature of mild, clinically homogeneous, recently diagnosed type II (Non-insulin-dependent) diabetics

SummaryAlteration in insulin secretion and reduced peripheral sensitivity to the hormone have been reported in type II diabetes. In this paper, a comparison is made of basal glucose production (3H-6 glucose), insulin secretion and insulin sensitivityin vivo (hyperglycemic clamp) andin vitro (binding to circulating monocytes) in 24 patients with recently diagnosed type II diabetes, matched for age and fasting glycemia and divided into non-obese (14 subjects) and moderately obese (10 subjects), and in 9 non-obese controls. The non-obese diabetics were slightly hyperinsulinemic during fasting (10.8±1.0vs 4.8±0.8 µU/ml in controls, p < 0.0005), with a significant reduction in early and late insulin secretion (14.0±1.5vs 20.8 ± 2.0 µU/ml, p<0.01 and 24.8±3.3vs 34.7±2.14 µU/ml, p<0.025). The insulin sensitivity index MCR/I was significantly reduced (2.30±0.32vs 4.14±0.40, p<0.005). Endogenous glucose production was significantly increased (107±10.2vs 84±3.7 mg/m2 per min, p<0.025) and displayed a positive correlation with fasting glycemia (r=0.51, p<0.05). Insulin binding to monocytes was significantly lower than in controls (2.36±0.22%vs 4.06±0.32%, p<0.0005). Moderately obese diabetics also were significantly hyperinsulinemic in the fasting state (18.1±2.8 µU/ml, p<0.0005vs controls) but, typically, lacked the early secretory phase (20.6±3.6 µU/mlvs baseline, n.s.). A similar increase of hepatic glucose production (107±11.2 mg/m2 per min, p<0.025vs controls, n.s.vs non-obese diabetics) and decrease of peripheral sensitivity to insulin (MCR/I=1.78±0.31, p<0.0005vs controls, n.s.vs non-obese diabetics) was found in moderately obsese diabetics, as well as a significant reduction of insulin binding to insolated monocytes (2.62±0.4% p<0.01vs controls, n.s.vs non-obese diabetics). These results confirm that common defects of both non-obese and moderately obese type II diabetics are: lack of early phase of glucose induced insulin secretion, increase in hepatic glucose production and decrease of peripheral insulin sensitivity together with reduction of insulin binding to circulating monocytes. The hypothesis of a unique defect as a cause of hyperglycemia in type II diabetes in early clinical phase is not borne out by the results of this study. Moderate obesity, even if able to reduce insulin sensitivity, seems to be less important in determining hyperglycemia.

Effects of sodium and potassium adenosine-triphosphatase on circulating lymphocytes: An approach to human obesity

SummaryThe methodological aspects of (Na+,K+)-ATPase-dependent uptake of86Rb, a potassium analog, were examined on human lymphocytes isolated from peripheral blood. The study of the time-course, the kinetic parameters, i.e., maximum velocity (Vmax) and Michaelis constant (Km) and the ouabain inhibition curve of86Rb+ uptake confirm that circulating lymphocytes represent a suitable model for the study of (Na+,K+)-ATPase in human diseases. An application to human obesity is reported: the results indicate that86Rb+ uptake on circulating lymphocytes is similar in obese and non-obese subjects. Therefore, (Na+,K+)-ATPase does not seem to be involved in the pathogenesis of human obesity.