Cell surface GLUT4 levels in skeletal muscle from nine t y p e 2 diabetic subjects and nine healthy control subjects have been assessed by a new technique that involves the use of a biotinylated photo-affinity label. A profound impairment in GLUT4 translocation to the skeletal muscle cell surface in response to insulin was observed in type 2 diabetic patients. Levels of insulinstimulated cell surface GLUT4 above basal in type 2 diabetic patients were only ~10% of those observed in healthy subjects. The magnitude of the defect in GLUT4 translocation in type 2 diabetic patients was greater than that observed for glucose transport activity, which was ~50% of that in healthy subjects. Reduced GLUT4 translocation is therefore a major contributor to the impaired glucose transport activity in skeletal muscle from type 2 diabetic subjects. When a marked impairment in GLUT4 translocation occurs, the contribution of other transporters to transport activity becomes apparent. In response to hypoxia, marked reductions in skeletal muscle cell surface GLUT4 levels were also observed in type 2 diabetic patients. Therefore, a defect in a common late stage in signal transduction and/or a direct impairment in the GLUT4 translocation process accounts for reduced glucose transport in t y p e 2 diabetic patients. D i a b e t e s 4 9 :6 4 7-654, 2000

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)

Insulin binding to its cell surface receptor activates a range of intracellular signalling cascades that ultimately result in the regulation of a number of important metabolic events within the cell [1]. Of particular importance is insulin stimulation of glucose transport and stimulation of glycogen deposition in muscle as this accounts for the bulk of insulin stimulated glucose disposal from the blood [2,3]. Peripheral insulin resistance at the level of skeletal muscle plays an important role in the development of hyperinsulinaemia and hyperglycaemia associated with obesity [4] and non-insulin-dependent diabetes [5]. The consensus of opinion is that defects in insulin signalling lead to the reduced ability of insulin to stimulate glucose disposal into muscle in insulin resistant individuals [6,7]. Understanding the molecular mechanisms by which insulin regulates glucose transport and glycogen storage in skeletal muscle is therefore of great importance. Insulin acutely stimulates phosphoinositide-3 (PI 3)-kinase and evidence suggests that PI 3-kinase activity is necessary [8][9][10] and sufficient [11,12] for insulin stimulation of glucose transport and also necessary for insulin activation of glycogen synthase [13,14]. Further, there is evidence to suggest that protein kinase B (PKB) is an element of the signalling cascade leading from PI 3-kinase to activation of glycogen synthase in cell culture models [15,16]. Insulin's regulation of these pathways has not been investigated in intact human skeletal muscle. Diabetologia (1997) 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 MAPkinase kinase (MEK) inhibitor PD 98059. However, PD98059 (up to 20 m mol/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 (IC 50 ≈ 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.

The 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.