The effect of insulin and insulin deficiency on the transport and metabolism of glucose by rat small intestine

1. Insulin deficiency induced by anti-insulin serum or streptozotocin increased glucose absorption, as measured in everted sacs of rat upper ileum incubated for 30 min with oxygenated Krebs-Henseleit bicarbonate medium.2. Everted sacs prepared from the terminal ileum of insulin-deficient rats were able to accumulate glucose against a concentration gradient (i.e. development of active glucose transport).3. In experimental diabetes induced by streptozotocin, everted sacs of upper ileum showed increased 3-methyl glucose active transport, and sacs of terminal ileum showed development of 3-methyl glucose active transport.4. Lactic acid formation during the absorption of both glucose and 3-methyl glucose was increased approximately twofold in everted sacs of insulin-deficient animals.5. Insulin added at 100 mu./ml. to the incubating media of everted sacs prepared from insulin-deficient rats did not result in a reduction of glucose absorption or reverse the other effects.6. Fluoride (5 x 10(-3)M) added to the serosal and mucosal media of sacs of terminal ileum prepared from insulin-deficient rats decreased [(14)C]CO(2) formation from [U-(14)C]glucose and lactate formation during glucose absorption, but was unable to reverse the effect of insulin deficiency on glucose active transport.7. The effects of insulin deficiency induced by streptozotocin were more striking than those induced by anti-insulin serum.8. Everted sacs prepared from rats starved for 3 days showed increased glucose active transport accompanied by diminished conversion of [U-(14)C]glucose to [(14)C]CO(2).9. The possible role of hexokinase is discussed in relation to these findings.     

Diabetes-induced decrease of adenosine kinase expression impairs the proliferation potential of diabetic rat T lymphocytes

The proliferative response of T lymphocytes is a crucial step in cell-mediated immunity. This study was undertaken to investigate the mechanisms leading to the impaired proliferative response of diabetic T lymphocytes. T cells that had been isolated from the spleen of normal rats and cultured in medium containing 20 mm glucose and no insulin displayed the same degree of proliferative impairment as cells isolated from diabetic rats. The rate of T-cell proliferation, when induced with concanavalin A or anti-CD3 and anti-CD28 antibodies, was not affected by the inhibition of nucleoside transporters. T cells cultured at high glucose concentrations in the absence of insulin displayed decreased expression of adenosine kinase, and released measurable extracellular quantities of adenosine. Under resting conditions, the level of cAMP was 5.9-fold higher in these cells compared to cells grown in low glucose and in the presence of insulin. Experiments with specific adenosine receptor agonists and antagonists showed that adenosine-induced suppression of diabetic T cell proliferation was mediated by the A2A adenosine receptor, but not by the A2B receptor. Treatment of diabetic T cells with 10 microm H-89, a specific protein kinase A inhibitor, restored T-cell proliferation. These results show that suppressed proliferation of diabetic T lymphocytes is evoked by the decreased expression of adenosine kinase, leading to the outflow of adenosine from the cell. Extracellular adenosine then stimulates the A2A receptor and induces cAMP production, leading to the activation of protein kinase A, and suppression of T-cell proliferation.     

Effects of insulin and 2-deoxy-D-glucose on plasma glucose level and lateral hypothalamic eating threshold in the rat

Thresholds for elicited eating through monopolar electrodes in the perifornical region of the lateral hypothalamus and plasma glucose concentration were determined over periods of several hours, while blood glucose levels and cellular glucose utilization were altered by means of forced feeding through hypothalamic stimulation, subcutaneous insulin injections and/or intraperitonneal 2-deoxy-D-glucose injections. Forced feeding resulted in increased thresholds for elicited eating, whereby the plasma glucose concentration in the tail vein was positively correlated to these thresholds. A subsequent, long-lasting, severe insulin-hypoglycemia was not sufficient to normalize such elevated thresholds. However, 2-deoxy-D-glucose in doses of 100–250 mg/kg, after an initial increase, decreased thresholds 90 min after injection. When insulin and 2-deoxy-D-glucose were combined so as to balance the contrary glycemic effects, the insulin effect dominated, resulting in slightly increasing thesholds. The results are discussed in terms of the recently suggested hypothesis that insulin regulates glucose entry into glucosensitive systems of the ventromedial hypothalamus, and thus generates satiety signals rather than hunger.     

Correlation between local glucose transporter densities and local 3-O-methylglucose transport in rat brain

The present study addresses the question whether local glucose transport kinetics are correlated with local glucose transporter densities in the brain. In 47 brain structures the local rate constants for 3-O-[¹⁴C]methylglucose (3-O-MG) transport, K₁ and k_2, were quantified, and local glucose Glut1 and Glut3 transporter densities were determined by immuno-autoradiographic methods. Statistically significant correlations were found between the rate constants for glucose transport and the transporter densities. The correlations were tighter for Glut1 than for Glut3. Inasmuch as 3-O-MG is transported by the same transporter as glucose, these results indicate that the local densities of glucose transporters determine local glucose transport rates in the brain.     

血管生成素-1激活tyrosine kinase/PI3K增加血管内皮细胞［Mg2+］i

目的：探讨血管生成素-1（Ang-1）对人脐静脉内皮细胞（HUVECs）内游离镁离子浓度（［Mg²⁺］_i）的调节机制。方法:我们采用荧光指示剂mag-fura-2，运用PTi 阳离子测定系统动态测HUVECs的［Mg²⁺］_i。结果:Ang-1诱导的［Mg²⁺］_i增加与细胞外Mg2+浓度无关。Ang-1诱导的［Mg²⁺］_i增加与细胞内Ca2+浓度无关。经酪氨酸激酶阻断剂(tyrphostin A23 和 genistein), 磷脂酰3激酶阻断剂 (wortmannin和LY294002)预处理，均显著阻断Ang-1诱导的［Mg²⁺］_i增加。但经活化丝裂原激活激酶阻断剂(SB202190和PD98059) 预处理，不能阻断Ang-1诱导的［Mg²⁺］_i增加。结论:Ang-1通过酪氨酸激酶/磷脂酰3激酶信号传递途径使细胞内的Mg2+库释放Mg2+，从而增加HUVECs的［Mg²⁺］_i。     

Protection of CD95-mediated apoptosis by activation of phosphatidylinositide 3-kinase and protein kinase B

Apoptosis may be triggered, in a variety of tissues, by interaction of the cell surface molecule CD95 with its specific ligand, CD95L. CD95 plays a physiological role in the regulation of the immune response; furthermore, alterations in CD95/CD95L function may contribute to the pathogenesis of a number of human diseases, including cancer, autoimmune diseases and viral infections. Many cells that express CD95, however, are not susceptible to CD95-mediated apoptosis. It is therefore important to identify the mechanisms that counteract the CD95 apoptotic process that are still poorly understood. Growth factors and lymphokines such as interleukin (IL)-4 that counteract CD95-mediated apoptosis may activate phosphatidylinositide 3-kinase (PI 3-kinase). We therefore used two different approaches to investigate the role of PI 3-kinase on CD95-mediated apoptosis. First we tested the effect of two pharmacological PI 3-kinase inhibitors, wortmannin and LY294002, on CD95 agonistic antibody-induced apoptosis in three different cell lines. Second, we co-expressed in COS7 cells CD95 with constitutively active PI 3-kinase. Results of both approaches indicate that active PI 3-kinase effectively protects against CD95-mediated apoptosis. Furthermore we extended our studies on the CD95 downstream mediator, FADD, and on the PI 3-kinase downstream mediator, the serine/threonine protein kinase PKB, using the co-expression approach in COS7 cells. We provide evidence that apoptosis induced by triggering the CD95 cell death receptor is counteracted by PI 3-kinase activation; moreover, PKB but not p70S6K represents the relevant downstream target of PI 3-kinase signaling.     

Wortmannin-mediated inhibition of phosphatidylinositol 3-kinase activity triggers apoptosis in normal and neoplastic B lymphocytes which are in cell cycle

The B cell functional response following ligation of surface(s) lgM is dependent upon the differentiation stage of the populationstudied: cross-linking slgM promotes proliferation of restingtonsillar follicular mantle (FM) B lymphocytes but induces apoptosisin the susceptible Epstein- Barr virus genome-negative Burkittlymphoma (BL) cell line Ramos (Ramos-BL). This study investigateswhether phosphatidylinositol-3-kinase (Pl3-kinase), which hasbeen reported to be intimately involved in the regulation ofcellular growth, plays a role in the regulation of these sig-promoted B cell responses, and uses the selective and irreversibleinhibitor of Pl3-kinase activity, wortmannin (Wm). In Ramos-BLB cells, at 8 h post-treatment, Wm triggers a transient increasein apoptosis of 16 ± 6.9% with a concomitant cellularloss of 16 ± 6.1% from the G₁ phase of cell cycle; [³H]thymidineincorporation also decreases by 33 ± 5.0%, from 37,274c.p.m. ± 10% to 25,127 c.p.m. ± 4.0%. Moreover,at 72 h culture, Wm inhibits anti-lgM-induced FM B lymphocytelevels of [³H]thymidine incorporation typically by 47% and triggers80% apoptosis from the G₀G₁ phase of cell cycle. Ramos-BL Bcells exhibit high basal levels of Pl3-kinase activity, as determinedby immunoprecipitation with antibody to the p85 regulatory subunitof Pl3-kinase and ³²P incorporation into phosphatidylinositol,which is not significantly affected by anti-lgM stimulation;by contrast, anti-lgM stimulates significant Pl3-kinase activityover negligible basal levels in FM B lymphocytes. Pre-treatmentwith Wm inhibits Pl3-kinase activity in both cell types. Takentogether these data indicate that in Ramos-BL B cells slgM-triggeredgrowth arrest and apoptosis is Pl3- kinase independent, whereasPl3-kinase activity is critical for slgM-triggered mitogenesisof FM B lymphocytes. Thus Pl3-kinase plays a pivotal role inthe regulation of both normal and neoplastic B lymphocyte progressionthrough the cell cycle, such that if this Pl3-kinase-dependentpathway is inhibited these cells default to apoptosis.     

Tyrosine kinase and cAMP-dependent protein kinase activities in CD40-activated human B lymphocytes

In vitro, human B lymphocytes undergo long-term proliferation when activated through CD40, a protein expressed on their cell surface. The nature of CD40-dependent signals in proliferating fresh human Epstein-Barr virus-negative B lymphocytes is currently unknown. In this study, a CD40-dependent B cell culture system was used to examine the role of different signal transduction elements. Protein kinase C (PKC) depletion generated by a long-term phorbol 12-myristate 13-acetate treatment had weak effects on proliferation. Rather, tyrosine phosphorylation was shown to be directly involved in mediating CD40-dependent signals. The use of the protein tyrosine kinase (PTK)-specific inhibitor herbimycin A dramatically decreased cellular proliferation without altering the activity of the human immunodeficiency virus-1 long terminal repeat (HIV-1 LTR), a promoter largely dependent on the binding of nuclear factor ϰB (NF-ϰB). In contrast, the cAMP-dependent protein kinase specific inhibitor H-89 totally inhibited HIV-1 LTR activity at a concentration as low as 100 nM without affecting cellular proliferation. Electrophoretic mobility shift assay (EMSA) and supershift assay using an NF-ϰB binding sequence from the ϰ light chain as a probe, revealed that both p65 (RelA) and c-Rel were present in CD40-stimulated B cells. While PKC depletion did not alter the NF-ϰB level, treatment of B lymphocytes with H-89 or herbimycin A provoked a decrease in the NF-ϰB level. These observations establish the importance of different signal transducing pathways leading to CD40 activation of B lymphocytes.     

Effects of dipyridamole on adenosine concentration,insulin sensitivity and glucose utilisation in soleus muscle of the rat

Adenosine has been shown to modulate the sensitivity of skeletal muscle to insulin (Budohoski et al. 1984). In an attempt to further characterize the modulatory action of adenosine on insulin sensitivity inskeletal muscle we have investigated the effect of the nucleoside transport inhibitor dipyridamole in isolated incubated soleus muscle strips. At a concentration of 50 M, dipyridamole increased the concentration of adenosine in the soleus muscle by 36% and in the incubation medium by 32%. At this concentration of dipyridamole, the basal rates (in the presence of 1 unit of insulin/ml) of lactate formation, 2-deoxy [2,6-³H]glucose phosphorylation and glucose oxidation were decreased by 48%, 43% and 47% respectively, whilst the rate of glycogen synthesis was unaffected. Insulin-stimulated rates (in the presence of 10000 unit of insulin/ml) of lactate formation, 2-deoxy [2,6-³H] glucose phosphorylation, glycogen synthesis and glucose oxidation were decreased by 70%, 30%, 26% and 20% respectively in the presence of 50 M dipyridamole. Although 50 M dipyridamole was required to exert a significant effect on medium and soleus muscle adenosine concentrations, statistically significant effects on glycolytic rate were observed at concentrations as low as 2 M dipyridamole.It is concluded that the results are not consistent with dipyridamole exerting an effect on skeletal muscle carbohydrate metabolism solely through elevation of the intracellular or interstial adenosine concentration, but strongly suggest that dipyridamole inhibits glucose transport and/or phosphorylation in skeletal muscle.     

Differential signaling through the Ig-α and Ig-β components of the B cell antigen receptor

The B cell antigen receptor is a complex containing the antigen-binding immunoglobulin molecules and the Ig-α/Ig-β heterodimer which presumably connects the B cell antigen receptor to intracellular signaling components. To analyze the functional properties of the cytoplasmic parts of the B cell antigen receptor, we used the K46 B lymphoma line (IgG2a, χ) to express chimeric molecules composed of the extracellular and transmembrane part of the CD8α molecule and the cytoplasmic sequence of either the Ig-α (CD8α/Ig-α), the Ig-β (CD8α/Ig-β) protein or the membrane-bound γ2a heavy chain (CD8α/γ2a). From these three types of chimeric molecules only CD8α/Ig-α and CD8α/Ig-β, but not CD8α/γ2a, could transduce signals, thus providing the first evidence that the cytoplasmic tail of Ig-α and Ig-β have a signaling capacity. After cross-linking with anti-CD8α antibodies, both molecules induced a similar increase in intracellular free calcium ion and in MAP kinase phosphorylation. Protein tyrosine kinases, however, were strongly activated via the CD8α/Ig-α and only marginally via the CD8α/Ig-β molecule. This suggests that the Ig-α and Ig-β proteins have distinct roles during signal transduction through the B cell antigen receptor.     

Correlation between local glucose transporter densities and local 3-O-methylglucose transport in rat brain

The present study addresses the question whether local glucose transport kinetics are correlated with local glucose transporter densities in the brain. In 47 brain structures the local rate constants for 3-O-[¹⁴C]methylglucose (3-O-MG) transport, K₁ and k_2, were quantified, and local glucose Glut1 and Glut3 transporter densities were determined by immuno-autoradiographic methods. Statistically significant correlations were found between the rate constants for glucose transport and the transporter densities. The correlations were tighter for Glut1 than for Glut3. Inasmuch as 3-O-MG is transported by the same transporter as glucose, these results indicate that the local densities of glucose transporters determine local glucose transport rates in the brain.     

Differential effect of anti-HLA class I monoclonal antibodies on resting and in vivo-induced B lymphocytes

The role of HLA class I antigens in B cell triggering was investigated by analyzing the effect of monoclonal antibodies (MAbs) directed to such molecules on the in vitro function of resting and in vivo-induced lymphoblastoid (LB) B cells. Staphylococcus aureus Cowan I (SAC)-induced proliferation of high-density B lymphocytes was markedly inhibited by W6/32 MAb (reactive with a monomorphic determinant contributed by an alpha-chain and beta 2-microglobulin) but not by FG2/2 MAb (reactive with beta 2-microglobulin). The inhibition was not due to either a toxic effect or a change in the response kinetics. In contrast, LB B cells' spontaneous DNA synthesis and IgG production was not altered by such MAb, although these cells possessed surface HLA class I antigens. These findings suggest that HLA class I molecules are involved in the activation process of resting but not mature B lymphocytes.     

PI3-Kinase-dependent electrogenic intestinal transport of glucose and amino acids

Intestinal glucose and amino acid transport is stimulated by the serum- and glucocorticoid-inducible kinase isoforms SGK1, SGK2, and SGK3 and protein kinase B which are, in turn, stimulated following activation of the phosphoinositol-3 kinase (PI3 kinase). The present study has been performed to explore whether pharmacological inhibition of the PI3 kinase affects electrogenic jejunal transport of glucose and amino acids. In Ussing chamber experiments, glucose (20 mM), phenylalanine (20 mM), glutamine (20 mM), cysteine (20 mM), and proline (20 mM) generated lumen negative currents (I _glc, I _phe, I _gln, I _cys, and I _pro), respectively, which gradually declined following application of the PI3 kinase inhibitor Wortmannin (1 μM). Within 40 min, Wortmannin treatment significantly decreased I _glc by 39 ± 10% (n = 5), I _phe by 70 ± 7% (n = 4), I _gln by 69 ± 8% (n = 4), I _cys by 67 ± 8% (n = 6), and I _prol by 79 ± 12% (n = 3). A similar decline of I _glc was observed following application of the PI3 kinase inhibitor LY294002 (50 μM). Exposure to the inhibitors did not significantly alter transepithelial potential difference and resistance in the absence of substrates for electrogenic transport. The observations suggest that the electrogenic transport of glucose and several amino acids requires the continued activity of PI3 kinase. R. Rexhepaj and F. Artunc shared first authorship.