Jorge Rincon

Leukocyte‐Cell Adhesion: A Molecular Process Fundamental in Leukocyte Physiology

Leukocyte-cell adhesion is a form of physical contact characterized by fast (firm) stickiness between the cells. To analyze the biology and molecular basis of this process, an adhesion-specific assay was developed: the phorbol ester-induced aggregation of human lymphocytes. This rapid and antigen-independent intercellular adhesion requires cellular metabolism, an intact cytoskeleton and extracellular divalent cations, and is mediated by preformed cell-surface proteins referred to as CAMs. Phorbol ester also induces aggregation of monocytes and granulocytes, as well as adhesion of T lymphocytes to either B cells or monocytes and of the leukocytes to vascular endothelial cells. By using the adhesion-specific assay and blocking monoclonal antibodies, several CAMs have been identified, namely the Leu-CAM family (CD11a-c/CD18) and ICAM-1 (CD54). The Leu-CAM family is composed of Leu-CAMa (CD11a/CD18), Leu-CAMb (CD11b/CD18) and Leu-CAMc (CD11c/CD18), three glycoprotein heterodimers made of a common beta-chain and distinct alpha-chains. ICAM-1 is an adhesive ligand for Leu-CAMa. Expression and use of the various CAMs is selective in different types of leukocytes. The Leu-CAMs have been purified and partially characterized. CD18, whose gene is on human chromosome 21, contains 5-6 N-linked complex-type oligosaccharides, and CD11 binds Ca++. Another adhesion pathway is mediated by CD2 and CD58. CD2, a glycoprotein selectively expressed by T cells, is a receptor for CD58, a cell-surface adhesive ligand with broad tissue distribution. Antibodies to the latter CAMs do not block the phorbol ester-induced lymphocyte aggregation. Adhesion is involved in a large variety of leukocyte functions. Anti-Leu-CAM antibodies block induction of IL-2 production and lymphocyte proliferation. Lymphocyte-mediated cytotoxicity is also inhibited. Endogenous NK and LAK cells use Leu-CAMs, ICAM-1 and CD2, and sometimes RGD receptors, to bind and kill tumor cells. Endogenous compounds such as H2O2 and LTB4 also induce Leu-CAM-dependent adhesion in monocytoid cells and granulocytes, respectively, and degranulation of the latter cells is enhanced by the adhesion process. Homologous CAMs have been identified in rabbit and mouse. In in vivo studies in the former species, anti-Leu-CAM antibodies block adhesion of leukocytes to vascular endothelium and thereby their migration into extravascular tissues. The antibodies thus inhibit granulocyte accumulation and plasma leakage in inflammatory lesions, and induce lympho- and granulocytosis, indicating that cell-adhesion contributes to the distribution of leukocytes in the body.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms Behind Insulin Resistance in Rat Skeletal Muscle After Oophorectomy and Additional Testosterone Treatment

The absence of female sex hormones, as well as testosterone treatment of oophorectomized (OVX) female rats has been demonstrated to result in decreased whole-body insulin-mediated glucose uptake. The cellular mechanism behind this insulin resistance and the role of low levels of female sex hormones as a risk factor for development of peripheral insulin resistance are not yet fully clarified. We assessed the protein expression of GLUT4 and glycogen synthase, as well as insulin-induced translocation of GLUT4 to the plasma membrane, in soleus skeletal muscle from control rats, OVX rats, and OVX rats treated for 8 weeks with testosterone (OVX + T). Whole-body insulin-mediated glucose uptake assessed by the hyperinsulinemic-euglycemic clamp procedure was 25% lower in OVX rats (P < 0.001) and addition of testosterone treatment further decreased insulin-mediated glucose uptake in OVX + T rats by 48% (P < 0.001) compared with controls. GLUT4 protein expression in soleus muscles was unaltered in the OVX and OVX + T rats compared with controls. Insulin induced a 3.7-fold increase (P < 0.05) in the plasma membrane content of GLUT4 in soleus muscle from control rats, whereas plasma membrane content of GLUT4 in soleus muscle from OVX or OVX + T rats was unaltered in response to insulin. Glycogen synthase protein expression in muscle homogenates was decreased by 25% in the OVX group (P < 0.05) and by 37% in the OVX + T group (P < 0.05) when compared with the control group. Insulin receptor and tyrosine kinase activities in the basal and insulin-stimulated states did not differ between the OVX and OVX + T rats. In conclusion, the absence of female sex hormones appears to decrease insulin-mediated whole-body glucose uptake via an impaired insulin-stimulated translocation of GLUT4 to the plasma membrane and by decreased protein expression of glycogen synthase. Testosterone treatment further impairs whole-body insulin-mediated glucose uptake, presumably by additional impairment of glycogen synthase expression.

Exercise in the Management of Non—Insulin-Dependent Diabetes Mellitus

SummaryThe incidence of non-insulin-dependent diabetes mellitus (NIDDM) has increased worldwide during the last decades, despite the development of effective drug therapy and improved clinical diagnoses. NIDDM is one of the major causes of disability and death due to the complications accompanying this disease. For the well-being of the patient, and from a public healthcare perspective, the development of effective intervention strategies is essential in order to reduce the incidence of NIDDM and its resulting complications. For the patient, and for society at large, early intervention programmes are beneficial, especially from a cost-benefit perspective. Physical activity exerts pronounced effects on substrate utilisation and insulin sensitivity, which in turn potentially lowers blood glucose and lipid levels. Exercise training also improves many other physiological and metabolic abnormalities that are associated with NIDDM such as lowering body fat, reducing blood pressure and normalising dyslipoproteinaemia. Clearly, regular physical activity plays an important role in the prevention and treatment of NIDDM.Since physical activity has been shown in prospective studies to protect against the development of NIDDM, physical training programmes suitable for individuals at risk for NIDDM should be incorporated into the medical care system to a greater extent. One general determinant in a strategy to develop a preventive programme for NIDDM is to establish a testing programme which includes VO2max determinations for individuals who are at risk of developing NIDDM.Before initiating regular physical training for people with NIDDM, a complete physical examination aimed at identifying any long term complications of diabetes is recommended. All individuals above the age of 35 years should perform an exercise stress test before engaging in an exercise programme which includes moderate to vigorously intense exercise. The stress test will identify individuals with previously undiagnosed ischaemic heart disease and abnormal blood pressure responses. It is important to diagnose proliferative retinopathy, microalbuminuria, peripheral and/or autonomic neuropathy in patients with NIDDM before they participate in an exercise programme. If any diabetic complications are present, the exercise protocol should be modified accordingly.The exercise programme should consist of moderate intensity aerobic exercise. Resistance training and high intensity exercises should only be performed by individuals without proliferative retinopathy or hypertension. Once enrolled in the exercise programme, the patient must be educated with regard to proper footwear and daily foot inspections. Fluid intake is of great importance when exercising for prolonged periods or in warm and humid environments. With the proper motivation and medical supervision, people with NIDDM can enjoy regular physical exercise as a means of enhancing metabolic control and improving insulin sensitivity.

Involvement of phosphoinositide 3-kinase in insulin stimulation of MAP-kinase and phosphorylation of protein kinase-B in human skeletal muscle: implications for glucose metabolism

Summary Isolated skeletal muscle from healthy individuals was used to evaluate the role of phosphoinositide 3-kinase (PI 3-kinase) in insulin signalling pathways regulating mitogen activated protein kinase (MAP-kinase) and protein kinase-B and to investigate whether MAP-kinase was involved in signalling pathways regulating glucose metabolism. Insulin stimulated glycogen synthase activity ( ≈ 1.7 fold), increased 3-o-methylglucose transport into human skeletal muscle strips ( ≈ 2 fold) and stimulated phosphorylation of the p42 ERK-2 isoform of MAP-kinase. This phosphorylation of p42 ERK2 was not blocked by the PI 3-kinase inhibitors LY294002 and wortmannin although it was blocked by the MAP-kinase kinase (MEK) inhibitor PD 98059. However, PD98059 (up to 20 μmol/l) did not block insulin activation of glycogen synthase or stimulation of 3-o-methylglucose transport. Wortmannin and LY294002 did block insulin stimulation of protein kinase-B (PKB) phosphorylation and stimulation of 3-o-methylglucose transport was inhibited by wortmannin (IC50≈ 100 nmol/l). These results indicate that MAP-kinase is activated by insulin in human skeletal muscle by a PI 3-kinase independent pathway. Furthermore this activation is not necessary for insulin stimulation of glucose transport or activation of glycogen synthase in this tissue. [Diabetologia (1997) 40: 1172–1177]

Hyperglycemia Activates Glucose Transport in Rat Skeletal Muscle Via a Ca2+-Dependent Mechanism

We investigated the acute effect of hyperglycemia on 3-O-methylglucose transport in isolated rat epitrochlearis muscles. High levels of glucose (20 mmol/1) induced an ∼ twofold increase in the rate of glucose transport when compared with muscles exposed to a low level of glucose (8 mmol/1) (P < 0.001). The hyperglycemic effect was additive to the effects of both insulin and exercise on the glucose transport rates. Dantrolene (25 αmol/l), a potent inhibitor of Ca2+ release from the sarcoplasmic reticulum, blocked the ability of hyperglycemia to increase glucose transport by 73% (P < 0.01). Although dantrolene had no effect on the non-insulin-stimulated or the insulin-stimulated glucose transport rates during normoglycemic conditions, the effect of exercise was completely blocked in the presence of dantrolene (P < 0.01). Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (500 nmol/l) had no effect on the activation of glucose transport by hyperglycemia, whereas the insulin-stimulated glucose transport was completely abolished (P < 0.001). These findings suggest that hyperglycemia activates glucose transport by a Ca2+- dependent pathway. The signaling system for this Ca2+- dependent activation of glucose transport does not involve the activation of PI 3-kinase and is separate from the mass-action effect of glucose on glucose transport.

Changes in glucose transport and protein kinase Cβ2 in rat skeletal muscle induced by hyperglycaemia

Aims/hypothesis. We have previously reported that hyperglycaemia activates glucose transport in skeletal muscle by a Ca2+-dependent pathway, which is distinct from the insulin-signalling pathway. The aim of this study was to explain the signalling mechanism by which hyperglycaemia autoregulates glucose transport in skeletal muscle. Methods. Isolated rat soleus muscle was incubated in the presence of various concentrations of glucose or 3-O-methylglucose and protein kinase C and phospholipase C inhibitors. Glucose transport activity, cell surface glucose transporter 1 and glucose transporter 4 content and protein kinase C translocation was determined. Results. High concentrations of 3-O-methylglucose led to a concentration-dependent increase in [3H]-3-O-methylglucose transport in soleus muscle. Dantrolene, an inhibitor of Ca2+ released from the sarcoplasmic reticulum, decreased the Vmax and the Km of the concentration-response curve. Protein kinase C inhibitors (H-7 and GF109203X) inhibited the stimulatory effect of high glucose concentrations on hexose transport, whereas glucose transport stimulated by insulin was unchanged. Incubation of muscle with glucose (25 mmol/l) and 3-O-methylglucose (25 mmol/l) led to a three fold gain in protein kinase Cβ2 in the total membrane fraction, whereas membrane content of protein kinase Cα, β1, δ, ɛ and ϑ were unchanged. A short-term increase in the extracellular glucose concentration did not change cell surface recruitment of glucose transporter 1 or glucose transporter 4, as assessed by exofacial photolabelling with [3H]-ATB-BMPA bis-mannose. Conclusion/interpretation. Protein kinase Cβ2 is involved in a glucose-sensitive, Ca2+-dependent signalling pathway, which is possibly involved in the regulation of glucose transport in skeletal muscle. This glucose-dependent increase in 3-0-methylglucose transport is independent of glucose transporter 4 and glucose transporter 1 translocation to the plasma membrane and may involve modifications of cell surface glucose transporter activity. [Diabetologia (1999) 42: 1071–1079]

H2O2 activates CD11b/CD18-dependent cell adhesion

Treatment of monoblastoid U-937 cells with low concentrations of H2O2 caused adhesion of the cells to plastic. The H2O2 induced adhesion was rapid with a t1/2 of congruent to 6 min and was optimally stimulated by 100 microM H2O2 with an ED50 of congruent to 50 microM. The response to H2O2 closely resembled the adhesive response of U-937 cells to phorbol esters in its time dependency, requirement for extracellular Mg2+ and inhibition by cytochalasin B as well as inhibition by monoclonal antibodies against the leucocyte adhesion molecules CD11b and CD18. Phorbol ester treatment of U-937 cells stimulated the phosphorylation of at least three endogenous substrates, pp28, pp34 and pp43, of which pp28 and pp43 also responded to H2O2-treatment with increased 32P-incorporation. The results suggest that H2O2 might be a physiological modulator of leucocyte adhesion, possibly operating by activating protein kinase C.

Effect of Insulin on GLUT4 Cell Surface Content and Turnover Rate in Human Skeletal Muscle as Measured by the Exofacial Bis-Mannose Photolabeling Technique

Insulin-stimulated glucose transport across the skeletal muscle cell membrane is a major regulatory step in postprandial glucose disposal. To estimate the total molar concentration of GLUT4 as well as the turnover rate of GLUT4 in human vastus lateralis muscles at the cell surface in the basal state and after insulin exposure, we have applied the sensitive exofacial bis-mannose photolabeling technique on in vitro incubated human skeletal muscle strips from healthy subjects. In addition, we have measured 3-O-methylglucose transport in other muscle strips prepared from the same surgically removed human skeletal muscle biopsies to compare glucose transport with cell surface level of GLUT4. Maximal in vitro insulin stimulation (2,400 pmol/l) resulted in a twofold increase compared with basal in both surface GLUT4 content (0.38 ± 0.05 vs. 0.19 ± 0.03 pmol/g wet muscle wt, P < 0.005) and 3-O-methylglucose transport (1.24 ± 0.13 vs. 0.63 ± 0.08 umol · ml−1 · h−1 P < 0.005). The insulin-induced increment in 3-O-methylglucose transport was strongly correlated with the insulin-induced increase in cell surface GLUT4 content (r2 = 0.91; P < 0.005). The calculated turnover rate of human skeletal muscle GLUT4 amounted to ∼8 × 104 min−1 at 35δC and was unaffected by insulin. In conclusion, maximal in vitro insulin stimulation of vastus lateralis muscle strips from healthy subjects resulted in a twofold rise in glucose transport as well as in cell surface content, whereas the turnover rate of GLUT4 was unaffected by insulin under the chosen experimental conditions.

Expression of Adhesion Molecules CD 11/CD 18 (Leu-CAMs, β2-Integrins), CD54 (ICAM-1) and CD58 (LFA-3) in B -Chronic Lymphocytic Leukemia

Cell adhesion molecules (CAMs) are cell surface proteins with unique specificities that allow intercellular adhesion. The importance of CAMs for normal lymphocyte growth and differentiation is underscored by the association between neoplastic disease states and abnormal CAM expression. In the present study we analysed the cell surface expression of several CAMs on peripheral blood lymphocytes from patients with progressive chronic lymphocytic leukemia of B-cell type (B-CLL) (n = 21) and stable monoclonal B-lymphocytosis of undetermined significance (B-MLUS) (n = 20). The CAM expression was analysed on the B-cell clone and on normal T- and NK-cell populations separately using monoclonal antibodies (MAbs). A phorbol ester-induced lymphocyte aggregation assay and blocking MAbs were also used. The B-cell clone in B-CLL expressed ICAM-1 (CD54) more frequently and at a higher density than in B-MLUS. The brightest CD54 expression was noted in patients with prominent lymphadenopathy and/or splenomegaly. The β2 in...