Glucose uptake in enterocytes: a test for molecular targets of okadaic acid

The main diarrheic shellfish poisoning (DSP) toxin is okadaic acid (OA). Although OA is a protein phosphatase 1 and 2A inhibitor less is known about the involvement of the toxin in diarrhea. The initial statement was that OA, by altering the phosphorylation state of proteins, might modify glucose uptake and consequently ionic and water reabsorption across the small intestine. This report presents studies of glucose transport in isolated rabbit enterocytes by using a fluorescent derivative of D-glucose. The dye allowed examining the relation between the toxic effect of OA and cellular mechanisms involved in glucose transport. The central findings are: (i) OA potentiates decrease on glucose uptake due to protein kinase A (PKA) inhibitors such as H89; and (ii) the increase of sugar uptake induced by the protein kinase C (PKC) inhibitor chelerythrine is enhanced by OA. Importance of this work is justified by the need to determine molecular targets of diarrheic toxins in intestinal cells.     

Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-³H]-glucose uptake was increased (57% above control levels, p < 0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p < 0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p < 0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process.     

High glucose inhibits fructose uptake in renal proximal tubule cells: involvement of cAMP, PLC/PKC, p44/42 MAPK, and cPLA2

The precise signal that regulates fructose transport in renal proximal tubule cells (PTCs) under high glucose conditions is not yet known although fructose has been recommended as a substitute for glucose in the diets of diabetic people. Thus, we investigated that effect of high glucose on fructose uptake and its signaling pathways in primary cultured rabbit renal PTCs. Glucose inhibited the fructose uptake in a time- and dose-dependent manner. A maximal inhibitory effect of glucose on fructose uptake was observed at 25 mM glucose after 48 h, while 25 mM mannitol and l-glucose did not affect fructose uptake. Indeed, 25 mM glucose for 48 h decreased GLUT5 protein level. Thus, the treatment of 25 mM glucose for 48 h was used for this study. Glucose-induced (25 mM) inhibition of fructose uptake was blocked by pertussis toxin (PTX), SQ-22536 (an adenylate cyclase inhibitor), and myristoylated amide 14-22 (a protein kinase A inhibitor). Indeed, 25 mM glucose increased the intracellular cAMP content. Furthermore, 25 mM glucose-induced inhibition of fructose uptake was prevented by neomycin or U-73122 (phospholipase C inhibitors) and staurosporine or bisindolylmaleimide I (protein kinase C inhibitors). In fact, 25 mM glucose increased the total PKC activity and translocation of PKC from the cytosolic to membrane fraction. In addition, PD 98059 (a p44/42 mitogen-activated protein kinase (MAPK) inhibitor) but not SB 203580 (a p38 MAPK inhibitor) and mepacrine or AACOCF3 (phospholipase A2 inhibitors) blocked 25 mM glucose-induced inhibition of fructose uptake. Results of Western blotting using the p44/42 MAPK and GLUT5 antibodies were consistent with the results of uptake experiments. In conclusion, high glucose inhibits the fructose uptake through cAMP, PLC/PKC, p44/42 MAPK, and cytosolic phospholipase A2 (cPLA2) pathways in the PTCs.     

Two triterpeniods from Cyclocarya paliurus (Batal) Iljinsk (Juglandaceae) promote glucose uptake in 3T3-L1 adipocytes: The relationship to AMPK activation

PurposeThe current study investigated the efficacy of Cyclocarya paliurus chloroform extract (CPEC) and its two specific triterpenoids (cyclocaric acid B and cyclocarioside H) on the regulation of glucose disposal and the underlying mechanisms in 3T3-L1 adipocytes.MethodsMice and adipocytes were stimulated by macrophages-derived conditioned medium (Mac-CM) to induce insulin resistance. CPEC was evaluated in mice for its ability by oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). To investigate the hypoglycemic mechanisms of CPEC and its two triterpenoids, glucose uptake, AMP-activated protein kinase (AMPK) activation, inhibitor of NF-κB kinase β (IKKβ) phosphorylation and insulin signaling transduction were detected in 3T3-L1 adipocytes using 2-NBDG uptake assay and Western blot analysis.ResultsMac-CM, an inflammatory stimulus which induced the glucose and insulin intolerance, increased phosphorylation of IKKβ, reduced glucose uptake and impaired insulin sensitivity. CPEC and two triterpenoids improved glucose consumption and increased AMPK phosphorylation under basal and inflammatory conditions. Moreover, CPEC and its two triterpenoids not only enhanced glucose uptake in an insulin-independent manner, but also restored insulin-mediated protein kinase B (Akt) phosphorylation by reducing the activation of IKKβ and regulating insulin receptor substrate-1 (IRS-1) serine/tyrosine phosphorylation. These beneficial effects were attenuated by AMPK inhibitor compound C, implying that the effects may be associated with AMPK activation.ConclusionsCPEC and its two triterpenoids promoted glucose uptake in the absence of insulin, as well as ameliorated IRS-1/PI3K/Akt pathway by inhibiting inflammation. These effects were related to the regulation of AMPK activity.     

Regulation of Na+-coupled glucose carrier SGLT1 by AMP-activated protein kinase

Abstract

AMP-activated protein kinase (AMPK), a serine/threonine kinase activated upon energy depletion, stimulates energy production and limits energy utilization. It has previously been shown to enhance cellular glucose uptake through the GLUT family of facilitative glucose transporters. The present study explored the possibility that AMPK may regulate Na⁺-coupled glucose transport through SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with and without AMPK and electrogenic glucose transport determined by dual electrode voltage clamping experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or expressing constitutively active ^γR70QAMPK (α1β1γ1(R70Q)) alone, the addition of glucose to the extracellular bath generated a current (I_g), which was half maximal (K_M) at ≈ 650 μM glucose concentration. Coexpression of ^γR70QAMPK did not affect K_M but significantly enhanced the maximal current (≈ 1.7 fold). Coexpression of wild type AMPK or the kinase dead ^αK45RAMPK mutant (α1(K45R)β1γ1) did not appreciably affect I_g. According to confocal microscopy and Western Blotting, AICAR (1 mM), phenformin (1 mM) and A-769662 (10 μM) enhanced the SGLT1 protein abundance in the cell membrane of Caco2 cells suggesting that AMPK activity may increase membrane translocation of SGLT1. These observations support a role for AMPK in the regulation of Na⁺-coupled glucose transport.     

Phosphorylation is Involved in the Regulation of the Taurine Influx Via the β-system in Ehrlich Ascites Tumor Cells

The role of 3′,5′-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases in the regulation of the taurine influx via the β-system in Ehrlich ascites tumor cells has been investigated. The taurine uptake by the β-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the β-system is stimulated by an increased level of cAMP or following addition of N⁶,2′-O-dibutyryl-3′,5′-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the β-system in the Ehrlich cells exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially stimulates taurine transport when the β-system is in State II. Received: 8 September/Revised: 9 November 1995     

Evaluation of therapeutic effect of Premna herbacea in diabetic rat and isoverbascoside against insulin resistance in L6 muscle cells through bioenergetics and stimulation of JNK and AKT/mTOR signaling cascade

BackgroundPremna herbacea Roxb., a perennial herb is well documented for its therapeutic uses among the traditional health care-givers of Assam, India. Scientific validation on the traditional use of the medicinal plant using modern technology may promote further research in health care.PurposeThis study evaluates the therapeutic potential of methanolic extract of P. herbacea (MEPH) against type 2 diabetes mellitus (T2DM) and its phytochemical(s) in ameliorating insulin resistance (IR), thereby endorsing the plant bioactives as effective anti-hyperglycemic agents.MethodsThe anti-diabetic potential of the plant extract was explored both in L6 muscle cells and high fructose high fat diet (HF-HFD) fed male Sprague Dawley (SD) rats. Bioactivity guided fractionation and isolation procedure yielded Verbascoside and Isoverbascoside (ISOVER) as bioactive and major phytochemicals in P. herbacea. The bioenergetics profile of bioactive ISOVER and its anti-hyperglycemic potential was validated in vitro by XFe24 analyzer, glucose uptake assay and intracellular ROS generation by flourometer, FACS and confocal microscopy. The potential of ISOVER was also checked by screening various protein markers via immunoblotting.ResultsMEPH enhanced glucose uptake in FFA-induced insulin resistant (IR) L6 muscle cells and decreased elevated blood glucose levels in HF-HFD fed rats. Isoverbascoside (ISOVER) was identified as most bioactive phytochemical for the first time from the plant in the Premna genus. ISOVER activated the protein kinase B/AMP-activated protein kinase signaling cascades and enhanced glucose uptake in IR-L6 muscle cells. ISOVER decreased the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) and increased that of mammalian target of rapamycin (mTOR), thereby attenuating IR. However, molecular docking revealed that ISOVER increases insulin sensitivity by targeting the JNK1 kinase as a competitive inhibitor rather than mTOR. These findings were further supported by the bioenergetics profile of ISOVER.ConclusionThis study for the first time depicts the functional properties of ISOVER, derived from Premna herbacea, in ameliorating IR. The phytochemical significantly altered IR with enhanced glucose uptake and inhibition of ROS through JNK-AKT/mTOR signaling which may pave the way for further research in T2DM therapeutics.     

Artemisia princeps Extract Promoted Glucose Uptake in Cultured L6 Muscle Cells via Glucose Transporter 4 Translocation

Artemisia princeps is a familiar plant as a food substance and medicinal herb. In this study, we evaluated the effects of an ethanol extract of A. princeps (APE) on glucose uptake in differentiated L6 muscle cells. Treatment with APE elevated deoxyglucose uptake, and translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane in L6 myotubes occurred. The PI3K inhibitor LY294002 attenuated glucose uptake induced by APE. Phosphorylation of the Ser⁴⁷³ residue of Akt was not observed, but phosphorylation of PI3K, Akt (Thr³⁰⁸), and atypical PKC was. In addition, APE stimulated phosphorylation of AMP-activated protein kinase (AMPK) at a level similar to 5′-amino-5-imidazolecarboxamide-riboside (AICAR). These results indicate that APE stimulates glucose uptake by inducing GLUT4 translocation, which is in part mediated by combination of the PI3K-dependent atypical PKC pathway and AMPK pathways.     

Factors affecting glucose uptake by the ruminal bacterium Bacteroides ruminicola

Summary Glucose uptake by whole cells of Bacteroides ruminicola B₁4 is constitutive. Potassium concentrations between 10 and 150 mm stimulated uptake over fourfold, while sodium had little effect on uptake. The involvement of potassium in glucose uptake by B. ruminicola was supported by strong inhibition of uptake by the ionophores valinomycin, lasalocid, and monensin. The electron transport inhibitor antimycin A had little effect on uptake, but menadione and acriflavine inhibited uptake by 30 and 48%, respectively. Potent inhibitors of uptake included oxygen, p-chloromercuribenzoate, HgCl₂, and o-phenanthroline. Sodium arsenate decreased uptake by 40%, suggesting that a high-energy phosphate compound and possibly a binding protein may be involved in glucose uptake. The protonophores carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol inhibited glucose uptake by 37 and 22%, respectively. Little change in uptake activity was observed at extracellular pH values between 4.0 and 8.0. Excess (10 mm) cellobiose, maltose, and sucrose inhibited glucose uptake less than 15%. High levels (0.15% w/v) of p-coumaric acid and vanillin decreased uptake by 32 and 37%, respectively, while 0.15% ferulic acid decreased uptake by 15%.     

UPTAKE OF ORGANIC SUBSTRATES BY CYCLOTELLA CRYPTICA (BACILLARIOPHYCEAE): EFFECTS OF IONS,IONOPHORES AND METABOLIC AND TRANSPORT INHIBITORS1,2

The uptake of glucose and amino acids by the euryhaline diatom Cyclotella cryptica Reimann, Lewin & Guillard does not appear to be related to proton gradients. Instead, the transport systems for these organic solutes show a strong requirement for the presence of NaCl. The relationship between uptake and NaCl concentration is hyperbolic, with optimal uptake rates being approached at 100 mM NaCl. High concentrations of KCl cause strong reductions in uptake rates. The (Na⁺, K⁺)-stimulated ATPase inhibitor ouabain has no effect on glucose uptake, whereas the diphenolic glucoside phlorizin and its aglucone phloretin are strongly inhibitory. The proton translocating uncoupler CCCP (carbonylcyanide m-chlorophenyl hydrazone) and the ATPase inhibitor DCCD (dicyclohexylcarbodiimide) both almost completely abolish glucose transport, and low concentrations of the ionophares monensin and valenomycin strongly inhibit glucose uptake by the diatom. The requirement of high external NaCl concentrations for glucose transport, and the inhibitory effect an transport of the Na⁺-specific ionophore monensin are consistent with a coupling of Na⁺ and organic substrate transport, but could also be explained by a Na⁺ requirement for glucose binding to a transport carrier, and/or a possible interference with energy producing reactions associated with a monensin-induced collapse of the normal Na⁺ gradient.     

Effects of ketamine on glucose uptake by glucose transporter type 3 expressed in Xenopus oocytes: The role of protein kinase C

We investigated the effects of ketamine on the type 3 facilitative glucose transporter (GLUT3), which plays a major role in glucose transport across the plasma membrane of neurons. Human-cloned GLUT3 was expressed in Xenopus oocytes by injection of GLUT3 mRNA. GLUT3-mediated glucose uptake was examined by measuring oocyte radioactivity following incubation with 2-deoxy-d-[1,2-³H]glucose. While ketamine and S(+)-ketamine significantly increased GLUT3-mediated glucose uptake, this effect was biphasic such that higher concentrations of ketamine inhibited glucose uptake. Ketamine (10 μM) significantly increased V_max but not K_m of GLUT3 for 2-deoxy-d-glucose. Although staurosporine (a protein kinase C inhibitor) increased glucose uptake, no additive or synergistic interactions were observed between staurosporine and racemic ketamine or S(+)-ketamine. Treatment with ketamine or S(+)-ketamine partially prevented GLUT3 inhibition by the protein kinase C activator phorbol-12-myrisate-13-acetate. Our results indicate that ketamine increases GLUT3 activity at clinically relevant doses through a mechanism involving PKC inhibition.     

Reciprocal inhibition of in vitro substrate movement into avian skeletal muscle

Plasma glucose and ketone concentrations are much higher in birds than in humans and birds exhibit resistance to insulin-mediated glucose uptake into muscle. Therefore, birds may offer a model in which to examine the effects of high plasma glucose and free fatty acid (FFA) concentrations on substrate preference. The present study examined the uptake of radiolabeled oleic acid (OA; C18:1) and radiolabeled glucose by skeletal muscle isolated from the forewing of English sparrows (Passer domesticus). In dose–response studies, unlabeled glucose and OA (20 mM each) inhibited the uptake of their respective radiolabeled counterparts. To examine the effects of glucose on OA uptake, muscles were incubated for 60 min in a buffer containing 20 mM glucose with the addition of radiolabeled OA. This level of glucose significantly decreased radiolabeled OA uptake by 36%. Using the same methodology, 20 mM OA significantly decreased radiolabeled glucose transport by 49%. Comparing control values for glucose (0.952 ± 0.04 μM/mg muscle) and OA uptake (2.20 ± 0.29 μM/mg muscle), it is evident that OA is preferentially taken up by avian skeletal muscle. As FFAs provide a greater amount of energy per mole (146 ATP/OA) than carbohydrates (36 ATP/glucose), storing and utilizing fats may be more energy-efficient for birds. As studies in mammals have shown that FFAs may impair glucose uptake pathways, it is suspected that high FFA uptake by avian skeletal muscle may induce their notably lower glucose transport.     

Ursolic Acid Increases Glucose Uptake through the PI3K Signaling Pathway in Adipocytes

Background

Ursolic acid (UA), a triterpenoid compound, is reported to have a glucose-lowering effect. However, the mechanisms are not fully understood. Adipose tissue is one of peripheral tissues that collectively control the circulating glucose levels.

Objective

The objective of the present study was to determine the effect and further the mechanism of action of UA in adipocytes.

Methods and Results

The 3T3-L1 preadipocytes were induced to differentiate and treated with different concentrations of UA. NBD-fluorescent glucose was used as the tracer to measure glucose uptake and Western blotting used to determine the expression and activity of proteins involved in glucose transport. It was found that 2.5, 5 and 10 µM of UA promoted glucose uptake in a dose-dependent manner (17%, 29% and 35%, respectively). 10 µM UA-induced glucose uptake with insulin stimulation was completely blocked by the phosphatidylinositol (PI) 3-kinase (PI3K) inhibitor wortmannin (1 µM), but not by SB203580 (10 µM), the inhibitor of mitogen-activated protein kinase (MAPK), or compound C (2.5 µM), the inhibitor of AMP-activated kinase (AMPK) inhibitor. Furthmore, the downstream protein activities of the PI3K pathway, phosphoinositide-dependent kinase (PDK) and phosphoinositide-dependent serine/threoninekinase (AKT) were increased by 10 µM of UA in the presence of insulin. Interestingly, the activity of AS160 and protein kinase C (PKC) and the expression of glucose transporter 4 (GLUT4) were stimulated by 10 µM of UA under either the basal or insulin-stimulated status. Moreover, the translocation of GLUT4 from cytoplasm to cell membrane was increased by UA but decreased when the PI3K inhibitor was applied.

Conclusions

Our results suggest that UA stimulates glucose uptake in 3T3-L1 adipocytes through the PI3K pathway, providing important information regarding the mechanism of action of UA for its anti-diabetic effect.     

Stimulation of cardiac glucose transport by inhibitors of oxidative phosphorylation.

Previously we showed that hypoxia in heart stimulates glucose transport via translocation of glucose transporters from intracellular membranes to the plasma membrane. We later showed that rotenone, an inhibitor of oxidative phosphorylation, also decreased intracellular transporters. Here, using another membrane fractionation technique, we show that rotenone increases plasma membrane transporters, and that another respiratory chain inhibitor, azide, acts similarly. Thus, they likely activate the same signaling pathway as hypoxia. Genistein, a tyrosine kinase inhibitor, inhibited insulin- and azide-stimulated 3-O-methylglucose transport similarly in cardiac myocytes. It also increased glucose transporters in the plasma membranes of perfused hearts even though it inhibited glucose uptake, suggesting effects on membrane trafficking. Another tyrosine kinase inhibitor, lavendustin A, and the cyclic nucleotide-dependent protein kinase inhibitors H-8 and H-7 had little effect on basal or azide-stimulated transport. Polymyxin B was a weak inhibitor of basal, insulin-stimulated, and azide-stimulated transport. A nitric oxide donor and a nitric oxide synthase inhibitor had no effect on basal and azide-stimulated transport. The results indicate that tyrosine kinases; protein kinases A, G, and C; and nitric oxide are not involved in the hypoxic activation of cardiac glucose transport.     

Eicosapentaenoic acid (20:5 n-3) increases fatty acid and glucose uptake in cultured human skeletal muscle cells

This study was conducted to evaluate the chronic effects of eicosapentaenoic acid (EPA) on fatty acid and glucose metabolism in human skeletal muscle cells. Uptake of [14C]oleate was increased >2-fold after preincubation of myotubes with 0.6 mM EPA for 24 h, and incorporation into various lipid classes showed that cellular triacylgycerol (TAG) and phospholipids were increased 2- to 3-fold compared with control cells. After exposure to oleic acid (OA), TAG was increased 2-fold. Insulin (100 nM) further increased the incorporation of [14C]oleate into all lipid classes for EPA-treated myotubes. Fatty acid beta-oxidation was unchanged, and complete oxidation (CO2) decreased in EPA-treated cells. Basal glucose transport and oxidation (CO2) were increased 2-fold after EPA, and insulin (100 nM) stimulated glucose transport and oxidation similarly in control and EPA-treated myotubes, whereas these responses to insulin were abolished after OA treatment. Lower concentrations of EPA (0.1 mM) also increased fatty acid and glucose uptake. CD36/FAT (fatty acid transporter) mRNA expression was increased after EPA and OA treatment compared with control cells. Moreover, GLUT1 expression was increased 2.5-fold by EPA, whereas GLUT4 expression was unchanged, and activities of the mitogen-activated protein kinase p38 and extracellular signal-regulated kinase were decreased after treatment with OA compared with EPA. Together, our data show that chronic exposure of myotubes to EPA promotes increased uptake and oxidation of glucose despite a markedly increased fatty acid uptake and synthesis of complex lipids.     

Effects of prostaglandin D2 on Na-dependent phosphate transport activity and its intracellular signaling mechanism in osteoblast-like cells

Inorganic phosphate (Pi) transport probably represents an important function of bone-forming cells in relation to extracellular matrix mineralization. In the present study, we investigated the effect of prostaglandin D₂ (PGD₂) on Pi transport activity and its intracellular signaling mechanism in MC3T3-E1 osteoblast-like cells. PGD₂ stimulated Na-dependent Pi uptake time- and dose-dependently in MC3T3-E1 cells during their proliferative phase. A protein kinase C (PKC) inhibitor calphostin C partially suppressed the stimulatory effect of PGD₂ on Pi uptake. The selective inhibitors of mitogen-activated protein (MAP) kinase pathways such as ERK, p38 and Jun kinases suppressed PGD₂-induced Pi uptake. The inhibitors of phosphatidylinositol (PI) 3-kinase and S₆ kinase reduced this effect of PGD₂, while Akt kinase inhibitor did not. These results suggest that PGD₂ stimulates Na-dependent Pi transport activity in the phase of proliferation of osteoblasts. The mechanisms responsible for this effect are activation of PKC, MAP kinases, PI 3-kinase and S₆ kinase.     

Thapsigargin and EGTA inhibit endothelin-1-induced glucose transport

We have previously demonstrated that ET-1 may enhance glucose transport in 3T3-L1 adipocytes, secondarily to its stimulatory effect on GLUT1 gene expression by a mitogen-activated protein kinase (MAPK)-dependent pathway. In the present study, we further tested the involvement of Ca²⁺ in glucose uptake in response to ET-1. Among a variety of Ca²⁺-related agents tested, EGTA and thapsigargin were found to suppress both the glucose uptake and intracellular Ca²⁺ mobilization induced by ET-1, as determined by Fura-2 analysis. However, a phospholipase C inhibitor, U73122, also eliminated the intracellular calcium mobilization induced by ET-1, but had no effect on ET-1-stimulated glucose uptake. The finding that neither EGTA nor thapsigargin had any influence on ET-1-induced MAPK activation implies that some mechanism downstream of MAPK activation is involved. Further investigation showed that both agents exerted global inhibitory effects on protein and RNA syntheses. Since both thapsigargin and EGTA may deplete endoplasmic reticulum (ER) Ca²⁺ stores, our results suggest that (1) ET-1-induced glucose transport is independent of ET-1's effect on Ca²⁺ mobilization and (2) depletion of ER Ca²⁺ stores per se may interfere with ET-1's effect on GLUT1 expression.     

Investigation of transport-associated phosphorylation of sugar in yeast mutants (snf3) lacking high-affinity glucose transport and in a mutant (fdp1) showing deficient regulation of initial sugar metabolism

Pool-labeling experiments with 2-deoxyglucose in derepressed cells of the yeastSaccharomyces cerevisiae confirmed the previously reported results pointing to the possible existence of transport-associated phosphorylation of sugar. In yeast mutants containing a disruption or an inactivating point mutation in thesnf3 gene, which codes for the high-affinity glucose carrier, no evidence for transport-associated phosphorylation of 2-deoxyglucose was observed. If transport-associated phosphorylation in yeast exists, it is apparently not mediated by the low-affinity glucose carrier. Mediation by the high-affinity carrier would fit with the known requirement of an active kinase for high-affinity sugar transport. A mixed type of uptake in cells having both carriers would explain many of the problems associated with the 2-deoxyglucose pool-labeling experiments. Since mutants that have only low-affinity glucose transport are not deficient in the glucose-induced RAS-mediated cAMP signal, transport-associated phosphorylation of glucose is not required for or involved in the induction of the signal. The yeastfdp mutant, which dies on media containing fermentable sugars because of overaccumulation of sugar phosphates, also did not show any evidence for the existence of transport-associated phosphorylation. The same was true for the double mutantfdp snf3. The latter also showed the typicalfdp phenotype, indicative that the lethality on media containing fermentable sugar is owing to aberrant regulation of low-affinity transport. The high protein kinase activity in thefdp mutant does not appear to be responsible for the absence of evidence for transport-associated phosphorylation, because another mutant with high protein kinase activity, thebcy mutant, displayed normal transport behavior.     

Protein phosphatase type 2A,PP2A,is involved in degradation of gp130

The interleukin-6 (IL-6) stimulates growth in cells such as multiple myeloma and B-cell plasmacytomas/hybridomas, while it inhibits growth in several myeloid leukemia cells. The IL-6 receptor has subunit called gp130. It was reported that Ser-782 of gp130 is phosphorylated by unidentified kinase(s) in cell extracts, and level of gp130 (S782A) transiently expressed on the cell surface of COS-7 is 6-times higher than that of the wild type. These results motivated us to analyze whether the phosphorylation of gp130 at Ser-782 is involved in its degradation or not. In this study, we demonstrated here that treatment of HepG2 cells with okadaic acid (OA), a potent inhibitor for PP2A, promotes phosphorylation of gp130 at Ser-782 and degradation of gp130. MG115, a proteasome inhibitor, suppressed this degradation. These effects of OA could not be replaced with tautomycetin (TC), an inhibitor for PP1. Purified PP2A dephosphorylated phospho-Ser-782 of gp130 in vitro. IL-6-induced activation of Stat3 was suppressed by preincubation of the cells with OA, suggesting that the IL-6 signaling pathway was blocked by OA through degradation of gp130. Taken together, present results strongly suggest that degradation of gp130 is regulated through a phosphorylation-dephosphorylation mechanism in which PP2A is crucially involved and that gp130 is a potential therapeutic target in cancers. (Mol Cell Biochem 269: 183–187, 2005)     

Glucose uptake by Cellulomonas fimi

Glucose uptake by whole-cell suspension of the facultative anaerobe Cellulomonas fimi, which was two-fold higher under aerobic conditions than under N₂ or H₂, was inhibited by inhibitors of electron transport and ATP synthesis and, particularly, by proton and metal ion ionophores. A variety of sugars, including 2-deoxyglucose, did not inhibit glucose uptake but cellobiose was a non-competitive inhibitor. Cells grown on cellobiose medium transported glucose at one half the rate of glucose-grown cells. Cellulomonas fimi has a highly specific active system for glucose transport.