Nerve growth factor and fibroblast growth factor influence post-fusion expression of Na-channels in cultured rat skeletal muscle

We have examined effects of nerve growth factor (NGF) and fibroblast growth factor (FGF) on the density of tetrodotoxin (TTX)-sensitive Na-channels in cultured rat skeletal muscle. Measurements were made of specific binding of [3H]saxitoxin (STX) and the frequency and rate of rise of spontaneously occurring action potentials, the physiological expression of Na-channel density. Cells were transferred to various growth conditions at 6 days in vitro, and measurements were made beginning 24 hr later. Both growth factors (GF) caused dose-related increases in Na-channels compared with myotubes maintained in normal, serum-supplemented growth medium. Maximum effects occurred with a concentration of NGF of 50 ng/ml and FGF of 15 ng/ml. Scatchard analysis of specific STX binding showed an increase in Bmax with no significant change in Kd. Similar increases occurred on rate of rise and frequency spontaneous action potential. Treatment of cultures with cycloheximide or actinomycin D, inhibitors of protein and RNA synthesis, completely prevented the increase in STX-binding induced by GF treatment. The results indicate that NGF and FGF have important effects on regulation of excitable cell gene products after differentiation.     

Effect of the antibodies to gangliosides on the Retzius neuron electrical activity and the functional activity of sodium channels for incoming current in leech

In the present study, the effect of antibodies to gangliosides on the Retzius neurons of the leech was investigated to study the spike activity and the functional activity of the Na-channels which generate the spike. A forty-minute incubation of the Retzius neurons in a 20% solution of antiganglioside serum in a Ringer solution provoked appearance of a double spike (a spike with two parts) connected with a decrease of the speed of the activation of the tetrodotoxin (TTX)-sensitive Nachannels. The high frequency synaptic activation of the neuron (10 Hz during 10 minutes) under the plasticity exchange of the gate system of the TTX-sensitive Na-channels. As a result of this, there was a disturbance of the habituation of the Retzius neuron to the high-frequency stimulation.     

Protein synthesis and secretion and RNA and DNA synthesis in human skin fibroblasts in a medium with a low serum content

Human skin fibroblasts, both postnatal and embryonic, were cultured in the stationary phase of growth for 6-10 days in the DMEM with bovine serum (BS), 0.1-0.5% fetal calf serum (FCS) or 1% human serum (HS). On the day 4 of culturing, a considerable increase was observed in the synthesis and secretion of protein by postnatal fibroblasts in the Eagle medium with 0.1-0.5% FCS, or with 0.5% BS, and in medium 199 with 0.1-0.5 BS, or with 0.1 FCS. Maximum synthesis and secretion of 14C-proline labeled protein was observed on day 2 of culturing of cells in the DMEM medium with 1% HS. In the DMEM medium with low serum content, protein synthesis being virtually unchanged, 75-80% of protein was secreted by cells into the culture medium with BS on days 2-4; in the medium with FCS such a high secretion of protein was observed only on day 4. High synthesis of protein by fetal fibroblasts in the DMEM medium with 0.1% BS and high protein secretion in all the media with 0.1% BS or 0.5% FCS were observed. The maximum level of secretion of protein by fibroblasts coincided with a considerable increase in both RNA and DNA syntheses. The data obtained suggest that cells in deep resting state actively react to the composition of the medium as well as to the quality and quantity of the serum. It may also be suggested that the mechanism of protein secretion has an important role in maintenance of the constant level of intracellular proteins in resting cells.     

Determinants of Deoxyglucose Uptake in Cultured Astrocytes: The Role of the Sodium Pump

Glucose utilization in primary cell cultures of mouse cerebral astrocytes was studied by measuring uptake of tracer concentrations of [3H]2-deoxyglucose ([3H]2-DG). The resting rate of glucose utilization, estimated at an extracellular K+ concentration ([K+]o) of 5.4 mM, was high (7.5 nmol glucose/mg protein/min) and was similar in morphologically undifferentiated and "differentiated" (dibutyryl cyclic AMP-pretreated) cultures. Resting uptake of [3H]2-DG was depressed by ouabain, by reducing [K+]o, and by cooling. These observations suggest that resting glucose utilization in astrocytes was dependent on sodium pump activity. Sodium pump-dependent uptake in 2-3-week-old cultures was about 50% of total [3H]2-DG uptake but this fraction declined with culture age from 1 to 5 weeks. Uptake was not affected by changes in extracellular bicarbonate concentration ([HCO3-]o) in the range of 5-50 mM but was significantly reduced in bicarbonate-free solution. At high [HCO3-]o (50 mM) uptake was insensitive to pH (pH 6-8), whereas at low [HCO3-]o (less than 5 mM) uptake was markedly pH-dependent. Elevation of [K+]o from 2.3 mM to 14.2-20 mM (corresponding to extremes of the physiological range of [K+]o) resulted in a 35-43% increase in [3H]2-DG uptake that was not affected by culture age or by morphological differentiation. Our results indicate a high apparent rate of glucose utilization in astrocytes. This rate is dynamically responsive to changes in extracellular K+ concentration in the physiological range and is partially dependent on sodium pump activity.     

Evaluation of serum‐free differentiation conditions for C2C12 myoblast cells assessed as to active tension generation capability

We have compared several serum‐free media for the differentiation of C2C12 myoblasts and assessed the extent of differentiation in several ways including as to active tension generation capability. C2C12 cells were allowed to differentiate in Dulbecco's modified Eagle's medium (DMEM) containing Ham's F‐12 (F‐12), AIM‐V (AIM), 0.2% Ultroser‐G in DMEM (Ult‐G), and 0.1% Sericin in DMEM (Sericin), compared with in DMEM supplemented with 2% horse serum (HS) or 2% calf serum (CS). C2C12 differentiation was assessed as the extent of myotube formation, glucose metabolism, protein expression, sarcomere formation, and active tension generation. All serum‐free media examined were capable of inducing myotube formation and the expression of muscle‐specific proteins. All serum‐free media except for F‐12 gave the sarcomere structure. Active tension generation was observed for cells that differentiated in AIM and Ult‐G, but the active tension generated by C2C12 cells that differentiated in Ult‐G was only ～25% in the case of myotubes that formed in HS. The addition of Ult‐G to the AIM resulted in improvement of the active tension generation capability, the active tension generated being ～3.4× compared to that in HS. The approach for assessing muscle cell differentiation presented in this study will be suitable for other studies that involve the differentiation of muscle cells. Biotechnol. Bioeng. 2010;107: 894–901. © 2010 Wiley Periodicals, Inc.     

Additive effect of islet amyloid polypeptide (IAPP/amylin) and insulin on 2-deoxyglucose uptake in mouse pancreatic acini

The effect of islet amyloid polypeptide (IAPP/amylin) on 2-deoxyglucose (2-DG) uptake was studied in isolated mouse pancreatic acini in the absence or presence of insulin. Synthetic rat IAPP-NH2 caused a dose-dependent stimulation of 2-DG uptake by mouse acini with a half-maximal concentration at 70 nM. The increase in 2-DG uptake by 1 microM IAPP-NH2 or 100 nM insulin was 68% or 60% above basal, respectively. In the presence of both 1 microM IAPP-NH2 and 100 nM insulin, the increase in 2-DG uptake was 145% above basal, indicating that the effects of IAPP-NH2 and insulin on 2-DG uptake were additive. The results suggest that IAPP stimulates glucose uptake in mouse acini probably by a different mechanism from that of insulin.     

The effect of trypsin on sugar uptake in rat thymocytes. Modulation of cellular cyclic AMP concentration and the sugar-transport system.

I have shown that cyclic AMP stimulates sugar uptake in rat thymocytes. However, trypsin treatment, which increases rat thymocyte cyclic AMP concentration, fails to increase sugar uptake. The purpose of the present study is to examine this seeming inconsistency, and to evaluate further the function of trypsin. Mild trypsin treatment of rat thymocytes produced a dose-related increase in cellular cyclic AMP concentration. Trypsin produced the same proportionate increase in cyclic AMP concentration in the presence or absence of optimal concentrations of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, which suggests that trypsin acts to increase thymocyte cyclic AMP concentration by stimulating adenylate cyclase activity. Trypsin at concentrations of 0.3 mg/ml and less had no effect on the uptake of the glucose analogue 2-deoxy-D-glucose (2-DG), whereas at concentrations of 1 mg/ml and higher trypsin produced a small, dose-related, decrease in basal 2-DG uptake, becoming significantly lower than control values only at 5 mg/ml (-22.7%, P less than 0.05). Thymocyte sugar transporters, characterized by means of cytochalasin B binding, consist of a single class of sites with an apparent KD of 0.15 microM and maximum binding capacity of 2.73 pmol/20 x 10(6) cells (8.4 x 10(4) sites/thymocyte). Trypsin produced a dose-related decrease in the sugar-displaceable binding of cytochalasin B, so that at 5 mg of trypsin/ml the number of sugar transporters was decreased by approx. 50%. Thus trypsin treatment of rat thymocytes on the one hand increases cellular cyclic AMP concentration, which itself potentiates 2-DG uptake, and on the other hand decreases the number of sugar transporters, which itself decreases cellular sugar uptake, indicating that the apparent effect of trypsin on thymocyte 2-DG uptake is the result of the balance of its effects on these two systems.     

PKC and MAPKs pathways mediate EGF-induced stimulation of 2-deoxyglucose uptake in mouse embryonic stem cells.

It has been reported that epidermal growth factor (EGF) and EGF receptor were highly expressed in embryo, suggesting that the EGF system is related to early embryo development in an autocrine and/or paracrine manner. Glucose becomes the preimplantation exogenous energy substrate and enters the blastocyst via glucose transporters. Thus, the effect of EGF on [3H]-2-deoxyglucose (2-DG) uptake and its related signaling pathways were examined in mouse embryonic stem (ES) cells. EGF significantly increased 2-DG uptake in time- and concentration- dependent manner (>12 hr, >10 ng/ ml) and increased mRNA and protein level of glucose transporter 1 (GLUT1) compared to control, respectively. Actinomycin D and cycloheximide completely blocked the effect of EGF on 2-DG uptake. EGF-induced increase of 2-DG uptake was blocked by AG1478 (EGF receptor tyrosine kinase blocker), genistein or herbimycin (tyrosine kinase inhibitors). In addition, EGF effect was blocked by neomycin and U 73122 [phospholipase C (PLC) inhibitors] as well as staurosporine and bisindolylmaleimide I [protein kinase C (PKC) inhibitors]. EGF was also observed to increase inositol phosphates (IPs) formation and activate a PKC translocation from the cytosolic to membrane fraction, suggesting a role of PLC and PKC. SB 203580 [p38 mitogen activated protein kinase (MAPK) inhibitor] or PD 98059 (p44/42 MAPKs inhibitor) blocked EGF-induced increase of 2-DG uptake. EGF also increased phosphorylation of p38 MAPK and p44/42 MAPKs, which was blocked by genistein or bisindolylmaleimide I, respectively. In conclusion, EGF partially increased 2-DG uptake via PKC, p38 MAPK, and p44/42 MAPKs in mouse ES cells.     

ANG II increases 2-deoxyglucose uptake in mouse embryonic stem cells

It is now suggested that all components of the renin-angiotensin system are present in many tissues, including the embryo and may play a major role in embryo development and differentiation. However, little is known regarding whether ANG II regulates glucose transport in mouse embryonic stem (ES) cells. Thus, the effects of ANG II on [3H]-2-deoxyglucose (2-DG) uptake and its related signal pathways were examined in mouse ES cells. ANG II significantly increased cell proliferation and 2-DG uptake in concentration- and time-dependent manner (>18 h, >10(-8) M) and increased mRNA and protein level of GLUT1 by 31+/-7% and 22+/-5% compared to control, respectively. Actinomycin D and cycloheximide completely blocked the effect of ANG II on 2-DG uptake. ANG II-induced increase of 2-DG uptake was blocked by losartan, an ANG II type 1 (AT1) receptor blocker, but not by PD 123319, an ANG II type 2 (AT2) receptor blocker. In addition, ANG II-induced stimulation of 2-DG uptake was attenuated by phospholipase C (PLC) inhibitors, neomycin and U 73122 and ANG II increased inositol phosphates (IPs) formation by 37+/-8% of control. Protein kinase C (PKC) inhibitors, staurosporine, bisindolylmaleimide I, and H-7 also blocked ANG II-induced stimulation of 2-DG uptake. Indeed, ANG II activated a PKC translocation from the cytosolic to membrane fraction, suggesting a role of PKC. A 23187 (Ca2+ ionophore) increased 2-DG uptake and nifedifine (L-type Ca2+ channel blocker) blocked it. In conclusion, ANG II increased 2-DG uptake by PKC activation via AT1 receptor in mouse ES cells.     

ACTH1-24 stimulates muscle cell glucose uptake

3H-2-deoxyglucose (2-DG) uptake was measured in L6A-1 rat skeletal muscle cells (a rapidly fusing subclone of L6), following addition of several concentrations (10(-16) to 10(-9)M) of the N-terminal fragment of ACTH1-24 to cells deprived of serum and insulin for 21 hours, but maintained in the presence of (5 micrograms/ml) insulin (stimulated state). There was a marked dose-dependent increase of 2-DG uptake at the various ACTH1-24 (P less than 0.001). There was no correlation between the time of exposure of the cells to serum-free conditions and the rate of uptake of 2-DG at the various ACTH1-24 concentrations both in the basal and insulin-stimulated states. Addition of catochalasin B (50 microM) to the cells, which inhibited both basal and insulin-stimulated uptake of 2-DG (by 70% and 91%, respectively) completely eliminated the enhancement of both of these uptake rates to 10(-12)M ACTH1-24. The results suggest that: 1) ACTH1-24 stimulates carrier-mediated uptake of glucose in skeletal muscle cells. 2) The site of action of ACTH1-24 is on the non-insulin mediated glucose uptake (NIMGU) system. 3) ACTH1-24 may be a useful probe to delineate some of the events associated with the NIMGU pathway.     

The beta2-adrenergic modulator celiprolol reduces insulin resistance in obese Zucker rats.

Essential hypertension is associated with an increased incidence of insulin resistance of skeletal muscle glucose transport. The present study determined if celiprolol, an antihypertensive agent with selective beta1-adrenoceptor antagonist and additional beta2-agonistic properties, administered by gavage either acutely (3 hr) or chronically (14 d), had a direct effect on improving glucose tolerance and insulin-stimulated glucose transport activity (using 2-deoxyglucose (2-DG) uptake) in isolated epitrochlearis muscles of the insulin-resistant obese Zucker rat. The effects of a selective beta1-blocker, metoprolol, were also assessed. Acute administration of celiprolol, but not metoprolol, increased insulin-stimulated 2-DG uptake in muscle by 22% (p<0.05). Chronic celiprolol treatment significantly lowered fasting plasma insulin (22%) and free fatty acids (40%) in comparison to obese control values. Moreover, chronic celiprolol administration decreased the glucose-insulin index (calculated as the product of the glucose and insulin areas under the curve during an oral glucose tolerance test), by 32% (p<0.05) compared to obese controls, indicating that peripheral insulin action was increased. Indeed, insulin-stimulated skeletal muscle 2-DG uptake was enhanced by 49% (p<0.05) in these celiprolol-treated obese animals. Metoprolol was without significant effect on any of these variables following chronic administration. These findings indicate that, in this animal model of insulin resistance, the beta1-antagonist/beta2-agonist celiprolol has a specific effect of improving insulin-stimulated skeletal muscle glucose transport that is independent of any hemodynamic alterations.     

人主动脉壁硫酸乙酰肝素蛋白聚糖对培养的人主动脉平滑肌细胞生长的影响

从动脉粥样硬化（ＡＳ）高（北京）、低（南宁）发区人正常胸主动脉内－中膜分离ＨＳＰＧ,观察其对体外培养的ＨＡＳＭＣ生长的影响,细胞计数、~３Ｈ－ＴｄＲ参入及形态观察均表明ＡＳ高、低发区人主动脉ＨＳＰＧ都能剂量依赖性地抑制ＨＡＳＭＣ增殖,但抑制百分数未见显著差异,结果提示,人动脉壁中ＨＳＰＧ的含量可能与ＡＳ发病有关．     

Oxidative stress stimulates skeletal muscle glucose uptake through a phosphatidylinositol 3-kinase-dependent pathway

We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum longus (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of H2O2, EDL muscles from rats were preincubated with the H2O2 scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment, but not SOD, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that H2O2 is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct H2O2 also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 micromol/l H2O2. H2O2-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not the nitric oxide inhibitor NG-monomethyl-l-arginine. H2O2 stimulated the phosphorylation of Akt Ser473 (7-fold) and Thr308 (2-fold) in isolated EDL muscles. H2O2 at 600 micromol/l had no effect on ATP concentrations and did not increase the activities of either the alpha1 or alpha2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism.     

Characteristics of Glucose Transport in Neuronal Cells and Astrocytes from Rat Brain in Primary Culture

Glucose transport systems in cultured neuronal cells and astrocytes of rats were characterized by measuring the uptake of 2-deoxy-D-[3H]glucose ([3H]2-DG) into the cells. Various sugars inhibited 2-DG uptake by neuronal cells and astrocytes similarly, a finding indicating that the substrate specificities of the transporters in the two types of cells were almost the same. However, the Km values for 2-DG of neuronal cells and astrocytes were 1.7 and 0.36 mM, respectively. The uptake of 2-DG was strongly inhibited by cytochalasin B. Nucleosides, such as adenosine, inosine, and uridine, inhibited 2-DG uptake competitively in both neuronal cells and astrocytes. The uptake by both types of cells were also inhibited by forskolin, but not by cyclic AMP, an observation suggesting that forskolin bound directly to the transporters to cause inhibition. Its inhibition was competitive in astrocytes and noncompetitive in neuronal cells. Astrocytes contained a glucose transporter with a subunit molecular weight of 45K, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a probe.     

Transport of 2-deoxy-d-[3H]glucose in microvessels isolated from bovine cerebral cortex

Microvessels were isolated from a bovine cortex and the transport of glucose was investigated by using 2-deoxy-d-[³H]glucose (2-DG). The apparentK _m for 2-DG transport was 118 M and therefore indicates a significant high affinity for the substrate. The inhibition of 2-DG uptake byd-glucose showed an apparentK _i of 222 M. Other sugars, e.g., 3-methyl-d-glucose andd-fructose, also inhibited the 2-DG uptake by 60.6 and 36.0%, respectively. Phloretin (1×10^–3 M) inhibited the 2-DG transport more than phlorizin (83.7 vs. 53.8%). Ouabain (1 and 5×10^–4 M) did not inhibit the uptake of 2-DG but 2,4-dinitrophenol (1×10^–4 M) did (78.0%). The uptake of 2-DG could not be demonstrated in homogenized microvessels. Adenine nucleotides (conc. 2 mM) had various effects on the 2-DG uptake by microvessels. ATP inhibited the uptake by 20.7%, ADP was virtually without effect, and AMP stimulated the uptake of 2-DG by 8.5%. It was also found that the decrease of adenylate energy charge favors the uptake of 2-DG. All these findings suggest that in cerebral microvessels of a bovine cortex, 2-DG is apparently transported by a specific, carrier-mediated transport system.Dedicated to Prof. Dr. R. Sammet on the occasion of his 60th birthday.     

Sympathetic activation of glucose utilization in brown adipose tissue in rats.

The effects of norepinephrine (NE) infusion and surgical denervation or electrical stimulation of the sympathetic nerves on 2-deoxyglucose (2-DG) uptake in interscapular brown adipose tissue (BAT) were investigated in vivo in rats to obtain direct evidence for sympathetic control of glucose utilization in this tissue. 2-DG uptake was rather low in fasted rats, but after refeeding it increased in the BAT as well as the heart, skeletal muscle, and white adipose tissue, in parallel with an increase in plasma insulin level. Cold exposure also enhanced 2-DG uptake in the BAT without the increase in plasma insulin level, while it had no appreciable effect on 2-DG uptake in other tissues. Sympathetic denervation greatly attenuated the stimulatory effect of cold exposure on 2-DG uptake in BAT, but it did not affect the increased 2-DG uptake after refeeding. Electrical stimulation of the sympathetic nerves entering BAT or NE infusion produced a marked increase in 2-DG uptake in BAT without noticeable effects in other tissues. beta-Adrenergic blockade, but not alpha-blockade, abolished the increased 2-DG uptake in BAT. It was concluded that glucose utilization in BAT is activated directly, independently of the action of insulin, by sympathetic nerves via the beta-adrenergic pathway.     

Leucine and mTORc1 act independently to regulate 2-deoxyglucose uptake in L6 myotubes

Chronic mTORc1 hyperactivation via obesity-induced hyperleucinaemia has been implicated in the development of insulin resistance, yet the direct impact of leucine on insulin-stimulated glucose uptake in muscle cells remains unclear. To address this, differentiated L6 myotubes were subjected to various compounds designed to either inhibit mTORc1 activity (rapamycin), blunt leucine intracellular import (BCH), or activate mTORc1 signalling (3BDO), prior to the determination of the uptake of the glucose analogue, 2-deoxyglucose (2-DG), in response to 1 mM insulin. In separate experiments, L6 myotubes were subject to various media concentrations of leucine (0–0.8 mM) for 24 h before 2-DG uptake in response to insulin was assessed. Both rapamycin and BCH blunted 2-DG uptake, irrespective of insulin administration, and this occurred in parallel with a decline in mTOR, 4E-BP1, and p70S6K phosphorylation status, but little effect on AKT phosphorylation. In contrast, reducing leucine media concentrations suppressed 2-DG uptake, both under insulin- and non-insulin-stimulated conditions, but did not alter the phosphorylation state of AKT-mTORc1 components examined. Unexpectedly, 3BDO failed to stimulate mTORc1 signalling, but, nonetheless, caused a significant increase in 2-DG uptake under non-insulin-stimulated conditions. Both leucine and mTORc1 influence glucose uptake in muscle cells independent of insulin administration, and this likely occurs via distinct but overlapping mechanisms.

     

Interleukin-6 gene expression is increased in insulin-resistant rat skeletal muscle following insulin stimulation

IL-6 expression in skeletal muscle is stimulated by contractions. We sought to examine whether hyperinsulinaemia increases IL-6 mRNA in skeletal muscle and whether any increase is modified in insulin resistant muscle. We hypothesized that intramuscular IL-6 mRNA would be increased in response to insulin, but such an affect would be unaffected by insulin resistance because the primary insulin sensitive signalling protein responsible for activating IL-6 functions normally in insulin resistant muscle. Transgenic rats over-expressing the gluconeogenic regulatory enzyme phosphoenolpyruvate carboxykinase (PEPCK) were studied. White gastrocnemius muscle samples were obtained under hyperinsulinaemic, euglycaemic clamp (4 mU kg(-1)min(-1) insulin, plasma glucose concentration 4-6 mmol L(-1)) and basal conditions in both PEPCK (basal n=4; insulin n=5) and wild-type (CON) (basal n=5; insulin n=4) rats, which were previously injected with a bolus of 2-[1-14C]deoxyglucose (2-DG) into the carotid artery. Muscle samples were assayed for 2-DG uptake and IL-6 mRNA. No differences in 2-DG uptake or IL-6 mRNA were observed when comparing groups under basal conditions. Under clamp conditions, 2-DG uptake was lower (P<0.05) in PEPCK compared with CON. Insulin stimulation in CON did not change IL-6 mRNA compared with basal levels. In contrast, there was an approximately 8-fold increase (P<0.05) in IL-6 mRNA in insulin-stimulated PEPCK compared with CON basal levels. Insulin stimulation increases IL-6 gene expression in insulin resistant, but not healthy, skeletal muscle, suggesting that IL-6 expression in skeletal muscle is sensitive to changes in insulin in circumstances of insulin resistance. It is likely that the differences observed when comparing healthy with insulin resistant muscle are due to the differential activation of insulin sensitive signalling proteins responsible for activating IL-6.     

Inhibition of T cell-mediated cytolysis by 2-deoxy-D-glucose (2-DG): differential effect of 2-DG on effector cells isolated early or late after alloantigenic stimulation in vitro.

The effect of the hexose analogue 2-deoxy-D-glucose (2-DG) on the functional activity of various populations of cytolytic T lymphocytes (CTL) has been compared. Under aerobic conditions, CTL harvested at the peak of the response (day 4) in primary or secondary mixed leukocyte cultures (MLC) were much more readily inhibited by 2-DG that CTL obtained from MLC at later times (day 11 to 18) or from the peritoneal cavity of alloimmune mice. Quantitatively, 0.4 mM 2-DG was sufficpient to inhibit cytolysis by 50% in day 4 CTL populatons, whereas 25 mM had little or no effect on day 11 to 18 CTL. Evidence was obtained that inhibition of cytolysis by 2-DG under these conditions was accompanied by a parallel inhibition of effector:target cell binding. In contradistinction to these findings, the cytolytic activity of both day 4 and day 11 MLC cells was readily inhibited by 2-DG under conditions where cell respiration was blocked by sodium azide. Furthermore, uptake of radiolabeled 2-DG was observed under aerobic conditions in both day 4 and day 11 MLC cells. These results strongly suggest that inhibition of cytolysis by 2-DG under aerobic conditions is mediated via a direct effect on CTL which is independent of the consequences of energy depletion. An indirect method by which CTL may be inhibited by 2-DG is suggested.     

Uniaxial cyclic stretch increases glucose uptake into C2C12 myotubes through a signaling pathway independent of insulin-like growth factor I

Insulin-like growth factor I (IGF-I), an autocrine/paracrine growth factor involved in myogenesis, has rapid effects on muscle metabolism. In a manner analogous to insulin and mechanical stimuli such as stretch, IGF-I stimulates glucose transport through recruitment of glucose transporters to surface membranes in skeletal muscles. It is known that IGF-I is secreted from skeletal muscle cells in response to stretch. Therefore, we examined whether IGF-I is involved in the mechanism by which mechanical stretch regulates glucose transport using cultured C2C12 myotubes. IGF-I increased 2-deoxy- D-glucose (2-DG) uptake, and this created an additive effect with mechanical stretch, suggesting that these stimuli enhance glucose transport through different mechanisms. In fact, IGF-I-stimulated 2-DG uptake was not blocked by dantrolene (an inhibitor of Ca (2+)release from sarcoplasmic reticulum), whereas the stretch-stimulated effect was abolished. Conversely, the IGF-I-stimulated 2-DG uptake was prevented by phosphatidylinositol 3-kinase inhibitor wortmannin, which did not prevent the stretch-stimulated 2-DG uptake. In addition, experiments using media conditioned by stretched myotubes indicated that a mechanically induced release of locally acting autocrine/paracrine growth factors was not sufficient for induction of 2-DG uptake. Thus, our results demonstrate that mechanical stretch signaling for glucose transport is independent of the mechanism through which IGF-I increases this transport.