The long-term effects of anti-retroviral protease inhibitors on sugar transport in L6 cells.

The objective of this study was to investigate the long-term effects of anti-retroviral protease inhibitors (PIs) on 2-deoxy-d -glucose (2-DG) transport in L6 cells in vitro. Exposure of L6 cells to saquinavir, ritonavir, indinavir and amprenavir resulted in significant increases in 2-DG transport using PI concentrations of 1-10 microM with continual exposure to PI. After removal of the PI for up to 48 h, 2-DG transport increases did not change and remained at pre-reversal levels. These changes in 2-DG transport were not related to stress-induced sugar transport or to apoptosis. The examination of glucose transporter (GLUT) 1, 3 or 4 translocation with subcellular fractionation indicated that insulin (i.e. 67 nM) could induce the translocation of all the GLUTs to the plasma membrane. Also, ritonavir (10 microM), which leads to a 2-fold increase in 2-DG transport, demonstrated increased GLUT (i.e. 1, 3 or 4) presence in the plasma membrane fraction, in the presence or absence of insulin. This increased 2-DG transport involved transporter presence in plasma membrane preparations and did not affect the ability of insulin to stimulate 2-DG transport with continual PI exposure. The mechanism(s) involved indicates ready reversibility of PI effects on transporters. The mechanism(s) why reversibility of PI-induced 2-DG transport was similar plus or minus PI was not apparent.

Effect of insulin-like growth factor I on HIV type 1 long terminal repeat-driven chloramphenicol acetyltransferase expression.

In this study, we have investigated the ability of insulin-like growth factor I (IGF-I) to inhibit HIV long terminal repeat (LTR)-driven gene expression. Using COS 7 cells cotransfected with tat and an HIV LTR linked to a chloramphenicol acetyltransferase (CAT) reporter, we observed that physiological levels of IGF-I (10(-9) M) significantly inhibited CAT expression in a concentration- and time-dependent manner. IGF-I did not inhibit CAT expression in COS 7 cells transfected with pSVCAT, and did not affect CAT expression in the absence of cotransfection with tat. Transfection of HIV-1 proviral DNA into COS 7 cells +/- IGF-I resulted in a significant decrease (p < 0.05) in infectious virion production. Both IGF-I and Ro24-7429 inhibited LTR-driven CAT expression, while TNF-alpha-enhanced CAT expression was not affected by IGF-I. On the other hand, a plasmid encoding parathyroid hormone-related peptide exhibited dramatic additivity of inhibition of CAT expression in COS 7 cells. Finally, we show that in Jurkat or U937 cells cotransfected with HIVLTRCAT/tat, IGF-I significantly inhibited CAT expression. Further, interleukin 4 showed in U937 cells inhibition of CAT expression that was not additive to IGF-I induced inhibition. Our data demonstrate that IGF-I can specifically inhibit HIVLTRCAT expression. This inhibition may occur at the level of the tat/TAR interaction. Finally, this IGF-I effect is seen in target cell lines and similar paths of inhibition may be involved in the various cell types employed.

Sugar transport and glut transporter expression in a variety of human immunodeficiency virus-1 (HIV-1) chronically infected target cell lines.

In this study, sugar transport and the cellular content of the human Glut 1 and 3 glucose transporters were ascertained in uninfected and chronically HIV-infected Jurkat and H9 cell lines (T-cell lines) and U937 cells (a promonocytic cell line). Sugar transport was determined by monitoring 2-deoxy glucose uptake (2DG) and glut transporter content was determined by Western analysis. Although 'acute' HIV infection of H9 cells led to increased cellular transport activity and Glut 3 transporter content, chronic HIV infection exhibited no significant differences in sugar transport in any of the cell types investigated whether log or stationary phase cultures were employed. When uninfected and chronically HIV-infected cell lines were compared, all cell lines expressed the Glut 1 transporter, however, significant differences in Glut 1 transporter content were not observed. The Glut 3 transporter which could only be detected in the H9 cell line exhibited no differences in Glut 3 content in uninfected or chronically HIV-infected cells (2.1 +/- 0.6 versus 3.8 +/- 2.1 x 10(-3) arbitrary units/microgram protein). A trend towards lower amino acid uptake was seen in the chronically HIV-infected cells but this was not significantly different from uninfected cell cultures. The data indicate that: (1) glucose transport and the Glut 1 and 3 transporters are not increased in cells chronically infected with HIV-1 and (2) the expression of the Glut 3 sugar transporters is not the same in all target cells.

Acylation-stimulating protein (ASP) regulates glucose transport in the rat L6 muscle cell line.

Acylation-stimulating protein (ASP), a human plasma protein, is a potent stimulator of triglyceride synthesis and glucose transport in both human adipocytes and fibroblasts. The purpose of the present in vitro study was to examine the effect of ASP on glucose transport in muscle cells. ASP stimulated 2-deoxy-glucose transport (2-DG) in differentiated rat L6 myotubes in a time (30 min to 24 h) and concentration dependent manner (97% increase). The magnitude of the ASP effect on glucose transport was comparable to the time- and concentration-dependent effects seen with insulin (125% increase), but was additive to insulin, pointing to involvement of differential signalling pathways. ASP stimulation was dependent on cell differentiation in that glucose transport increased by only 12% in myoblasts, comparable to the effect of insulin in myoblasts (15% increase) demonstrating selective responsiveness of the differentiated myotubes to ASP and insulin. The mechanism for the ASP induced increase in glucose transport was also examined. ASP increased the Vmax for 2-DG transport by 183% (4.02 vs. 1.42 nmol/mg cell protein/30 s; ASP vs. Control, respectively). This could be explained by an increased translocation of glucose transporters (GLUT 1, GLUT 4 and GLUT 3) to the plasma membrane surface as demonstrated by Western analysis (+43% P < 0.05, +30% P < 0.05, and +49% P < 0.05, respectively). The effects of ASP were equal to those of insulin (+47%, +26% and +53% for GLUT 1, GLUT 4 and GLUT 3, respectively) and in all cases were paralleled by comparable glucose transport increases under the same incubation conditions. After long-term stimulation (24 h), Western analysis indicated that ASP had a permissive effect on insulin stimulated increases in total GLUT3 and GLUT4 cellular transporter content. These results suggest that muscle is also responsive to ASP and that ASP may play a role in glucose metabolism in both muscle and adipose tissue.

Evidence that modulation of glucose transporter intrinsic activity is the mechanism involved in the allose-mediated depression of hexose transport in mammalian cells.

In serum starved V79 Chinese hamster lung fibroblast cells, replacement of D-glucose with D-allose resulted in a significant 38 +/- 18% (P < 0.05) reduction of 2-deoxy-D-glucose (2-DG) transport. Similarly, in a respiration-deficient mutant cell line (V79-G14), which has elevated 2-DG transport activity, D-allose reduced 2-DG transport by 59 +/- 18% (P < 0.05). [3H]D-allose uptake by V79 cells occurred slowly and was not inhibited by cytochalasin B, suggesting diffusion as the mode of D-allose entry. Western blot analysis using a rabbit polyclonal antibody to the human erythrocyte glucose transporter (GT) demonstrated that, in both cell lines, GT content and GT subcellular distribution were not significantly different in D-glucose vs. D-allose-treated cells. delta-Antibody, which has been shown to bind to exofacial epitopes of the GT (Harrison et al., 1990, J. Biol. Chem., 265:5793-5801), did not demonstrate any differences in surface binding to D-glucose vs. D-allose-treated intact V79 cells. D-allose treatment of 3T3 fibroblasts resulted in a similar decrease (72%) of 2-DG transport, however D-allose had no apparent effect on basal sugar transport in 3T3 adipocytes. These results suggest that D-allose reduces sugar transport through a modulation of the intrinsic activity of the GT, and that D-allose may act in a tissue-specific manner.

An improved technique for the isolation of lymphocytes from small volumes of peripheral mouse blood.

Centrifugation of mouse blood on density gradients of polyvinylpyrrolidone-coated colloidal silica (Percoll) resulted in the simultaneous separation of platelets, lymphocytes, granulocytes and erythrocytes. By this method, we have been successful in isolating a fraction of highly purified and viable lymphocytes from small volumes of peripheral mouse blood with good recovery.

Studies of cellular sensitization to myelin antigens in multiple sclerosis. Dissociation of MIF and LBT production in response to a peptide encephalitogenic in rhesus monkeys.

The macrophage migration inhibitory factor (MIF) assay and the lymphoblastic transformation (LBT) technique were utilized simultaneously to measure immune responses to peptide Y, the 17 amino acid C-terminal fragment of basic myelin protein, in patients with multiple sclerosis (MS). Ten normals and 67 MS patients from the Montreal Neurological Hospital and affiliated institutions were examined. A prospective attempt was made to correlate the measured responses with phasic clinical activity of the disease. The LBT results indicate some degree of cellular sensitization to peptide Y which parallels the clinical course of the illness, and resembles earlier positive findings obtained with the whole basic myelin protein molecule. These findings, however, are in contrast to a negative MIF response to the Y peptide used in the present study and further contrast the positive MIF results obtained earlier using the whole protein. It is not evident from the results of the present study whether sensitization may be of any pathogenetic significance, but the findings show that differing portions of the basic myelin protein molecule may selectively stimulate specific lymphokine elaboration by sensitized lymphocytes.