Stimulation of hexose transport in cultured human skin fibroblasts by insulin.

The effect of insulin on hexose transport in cultured human skin fibroblasts. Studies were carried out on cultures of human skin fibroblasts to explore the effect of insulin on hexose transport in serum-starved monolayers. Insulin (100 mU/ml) stimulated 2-deoxy-D-glucose transport (30% above control values) after 30 minutes exposure time, the response being similar up to four hours exposure to insulin. In several experiments (n = 22) employing three cell strains, insulin (100 mU/ml) exposure led to variable stimulation of 2-deoxy-D-glucose transport (an average of 37% above control values, with a range of 0 = 120%). The insulin-induced stimulation of 2-deoxy-D-glucose transport showed a dose dependency with increasing amounts of insulin, the response being maximal at an insulin concentration of 100 mU/ml. Kinetic analysis of 2-deoxy-D-glucose transport showed that insulin addition resulted in a slight change in the transport K_m (3.13 to 4.06 mM) and a 1.8-fold increase in the transport V_max (17.6 nanomoles/mg protein/min to 32.1 nanomoles/mg protein/min). Insulin also stimulated the transport of 3-0-methyl-D-glucose while the hexokinase activity of the cells was not affected. Further, this insulin-induced stimulation of sugar transport was not blocked by cycloheximide. The results indicate that insulin stimulated the stereospecific carrier-mediated of hexose transport in cultured human skin fibroblasts.     

Effects of lysine-vasopressin (LVP) and 1-deamino-8-D-arginine-vasopressin (dDAVP) upon electrical potential, short-circuit current and transepithelial D.C. resistance of the frog skin

The synthetic analogue of vasopressin, 1-deamino-8-D-arginine-vasopressin (dDAVP), possesses a protracted antidiuretic activity while having practically no pressoric activity as compared to arginine-vasopressin (AVP) or lysine-vasopressin (LVP). The effects of LVP and dDAVP were studied on the frog skin (Rana temporaria) sodium transport as reflected by the short-circuit current (SCC) level, on an Ussing apparatus. The application two different equimolar doses of LVP or dDAVP (approx. 9.4 X 10(-8) mol X l-1 and 18.8 X 10(-8) mol X l-1 to the inner surface of the skin resulted in identical maximal increases of sodium transport. However, the maximum transport stimulation after the application of dDAVP was delayed by about 30 min as compared to the stimulation by LVP (P less than 0.01). In addition, a protracted recovery of SCC towards its original levels was observed in experiments with dDAVP application after the hormone removal (P less than 0.01). It is concluded that dDAVP stimulates Na+ transport through the frog skin despite its lacking pressoric activity. Thus, the natriferic activity of vasopressin is related to its antidiuretic rather than pressoric activity. Maximum increase in the sodium transport following dDAVP application was delayed and more protracted as compared to the effect of LVP.     

The in vivo electrical parameters of toad skin

Open-circuit voltage (PD) and short-circuit current (SCC) across toad skin were studied in in vivo conditions. An improved technique for fastening a lucite chamber on the abdominal region of the animal was developed. Saline bridges (230 mM NaCl in 4% agar solution) were placed subcutaneously to make the connections between the extracellular fluid and the half-cells. A clear relationship was observed between the electrical parameters and sodium transport by the skin, since PD and SCC were related to the sodium concentration of the bathing solution, and abolished by the presence of amiloride--a specific sodium transport inhibitor in epithelia. The initial control values of SCC in vivo were higher than those in vitro, which was attributed to hormonal stimulation. However, these high initial control values of SCC in vivo fell with time, reaching steady levels after a 2 hr period. Vasopressin failed to increase SCC in vivo when the external sodium concentration was 115 mM, being effective only when the sodium concentration was low (5 mM). On the other hand, in isolated preparations vasopressin significantly promoted an increase in both PD and SCC.     

The hormone-sensitive hepatic Na+-pump. Evidence for regulation by diacylglycerol and tumor promoters

Ouabain-sensitive 86Rb+ uptake by isolated rat hepatocytes was studied to elucidate how Ca2+-mobilizing hormones stimulate the Na+-pump. Stimulation of this uptake was observed with concentrations of vasopressin ([8-arginine]vasopressin, AVP), angiotensin II, and norepinephrine which elicited Ca2+ mobilization and phosphorylase activation. These results suggested that changes in cytosolic Ca2+, mediated by inositol trisphosphate, might trigger sodium pump stimulation by AVP. However, in hepatocytes incubated in Ca2+-free Krebs-Henseleit buffer, Na+-pump activity was not altered over 15 min by either 1.5 mM EGTA or 1.5 mM Ca2+. Furthermore, incubation of cells in 5 mM EGTA for 15-30 min drastically impaired the ability of AVP to increase cytosolic Ca2+, but only modestly attenuated AVP-stimulated Na+-pump activity. Two tumor promoters, phorbol myristate acetate (PMA) and mezerein, stimulated Na+/K+-ATPase-mediated transport activity. Similarly, addition of synthetic diacylglycerols or of exogenous phospholipase C from Clostridium perfringens to increase endogenous diacylglycerol levels also resulted in a stimulation of the Na+-pump in the absence of changes in cytosolic or total cellular Ca2+ levels. Stimulation of the Na+-pump by the combination of maximal concentrations of PMA and AVP did not produce an additive response, and both agents displayed a transient time course, suggesting that the two agents share a common mechanism. Stimulation of the Na+-pump by AVP and PMA was not blocked by amiloride analogs which inhibit Na+/H+ exchange, but these compounds blocked the action of insulin. These data suggest that the elevated Na+/K+-ATPase-mediated transport activity observed in hepatocytes following exposure to Ca2+-mobilizing hormones is a consequence of stimulated diacylglycerol formation and may involve protein kinase C.     

Effect of phenylarsine oxide on fluid phase endocytosis: further evidence for activation of the glucose transporter

We have shown previously that insulin stimulates fluid phase endocytosis in 3T3-L1 adipocytes (Gibbs et al., 1986). Using [14C]sucrose as an endocytotic marker, we show here that phenylarsine oxide, a trivalent arsenical which binds neighboring dithiols, blocked not only insulin-stimulated fluid phase endocytosis, but basal endocytosis as well. The Ki for this process was 6 microM in the presence or absence of insulin and the time required for inhibition was less than 2.5 min, the limit of detection in our assay system. These results can be compared with the inhibitory effect of phenylarsine oxide on insulin-stimulated glucose transport. Although the Ki for insulin-stimulated transport (7 microM) was similar to that for inhibition of endocytosis, basal glucose transport was not affected by the inhibitor. Further, when cells were prestimulated with insulin causing maximal stimulation of the glucose transport rate, phenylarsine oxide induced a time-dependent reduction to the basal rate (t 1/2 of 10 min), despite the fact that endocytosis was blocked immediately. This observation suggests that if the transporter is recycled by an exocytotic/endocytotic mechanism, it is distinct from fluid-phase endocytosis/exocytosis, which is a vesicle-mediated process, and provides further evidence that the transporter may undergo intrinsic activation/inactivation which does not require vesicle movement.     

How muscle insulin sensitivity is regulated: testing of a hypothesis

Muscle contractions induce an increase in glucose transport. The acute effect of muscle contractions on glucose transport is independent of insulin and reverses rapidly after cessation of exercise. As the acute increase in glucose transport reverses, a marked increase in the sensitivity of muscle to insulin occurs. The mechanism for this phenomenon is unknown. We hypothesize that an increase in insulin sensitivity is a general phenomenon that occurs during reversal of an increase in cell surface GLUT4 induced by any stimulus, not just exercise. To test this hypothesis, epitrochlearis, rat soleus, and flexor digitorum brevis muscles were incubated for 30 min with a maximally effective insulin concentration (1.0 mU/ml). Muscles were allowed to recover for 3 h in the absence of insulin. Muscles were then exposed to 60 microU/ml insulin for 30 min followed by measurement of glucose transport. Preincubation with 1.0 mU/ml insulin resulted in an approximately 2-fold greater increase in glucose transport 3.5 h later in response to 60 microU/ml insulin than that which occurred in control muscles treated with 60 microU/ml insulin. Pretreatment of muscles with combined maximal insulin and exercise stimuli greatly amplified the increase in insulin sensitivity. The increases in glucose transport were paralleled by increases in cell surface GLUT4. We conclude that stimulation of glucose transport by any agent is followed by an increase in sensitivity of glucose transport to activation that is mediated by translocation of more GLUT4 to the cell surface.     

Inhibitory effect of sodium ursodeoxycholate on basal and stimulated short-circuit current across the isolated toad skin

The effect of sodium ursodeoxycholate (U) on short-circuit current (SCC), an index of basal and stimulated net ion transport across isolated skins of Bufo arenarum toads, was tested. U inhibited basal SCC when added to the epidermal side of the skins. The inhibitory effect was reversible after rinsing the preparation during 60 min. U also inhibited the natriferic response to oxytocin, db-cAMP and theophylline by 82%, 49% and 47%, respectively. Inhibition of SCC by exposure to U was reversed by the polyene antibiotic nystatin. In turn, SCC induced by nystatin in the amiloride-treated skin was insensitive to U and blocked by ouabain, a Na⁺, K⁺-ATPase inhibitor. These results strongly suggest that the effect of U is exerted at the apical membrane of sodium transporting cells, and rule out the existence of an additional site of inhibitory action of U.     

Activation of glucose transport in skeletal muscle by phospholipase C and phorbol ester. Evaluation of the regulatory roles of protein kinase C and calcium

It has been hypothesized on the basis of studies on BC3H-1 myocytes that diacylglycerol generation with activation of protein kinase C (PKC) is involved in the stimulation of glucose transport in muscle by insulin (Standaert, M. L., Farese, R. V., Cooper, R. D., and Pollet, R. J. (1988) J. Biol. Chem. 263, 8696-8705). In the present study, we used the rat epitrochlearis muscle to evaluate the possibility that PKC activity mediates the stimulation of glucose transport by insulin in mammalian skeletal muscle. Phospholipase C from Clostridium perfringens (PLC-Cp), which generates diacylglycerol from membrane phospholipids, and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) induced increases in glucose transport activity (assessed using 3-O-methylglucose transport) that were approximately 80 and approximately 20% as great, respectively, as that induced by a maximal insulin stimulus. PLC-Cp and PMA both caused a approximately 2-fold increase in membrane-associated PKC activity. In contrast, insulin did not affect PKC activity. These findings argue against a role of diacylglycerol-mediated PKC activation in the stimulation of skeletal muscle glucose transport by insulin. They also show that the BC3H-1 myocyte is not a good model for studying regulation of glucose transport in skeletal muscle. Neither the submaximal nor maximal effects of PLC-Cp and insulin on glucose transport were additive, suggesting that PLC-Cp interferes with insulin action. The maximal effects of PLC-Cp and hypoxia or muscle contractions were also not additive. However, the submaximal effects of hypoxia and PLC-Cp were completely additive. These findings raise the possibility that PLC-Cp stimulates glucose transport by the exercise/hypoxia-activated, not the insulin-activated, pathway in skeletal muscle. Exposure to PLC-Cp activated glycogen phosphorylase and potentiated twitch tension in response to electrical stimulation, providing evidence that PLC-Cp increases cytoplasmic Ca2+ concentration. Dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, completely blocked both the activation of phosphorylase and the stimulation of glucose transport by PLC-Cp. These findings provide evidence that an increase in cytoplasmic Ca2+ concentration is involved in the activation of glucose transport in skeletal muscle by PLC-Cp.     

Activation of p38 MAP kinase enhances sensitivity of muscle glucose transport to insulin

Muscle contractile activity is followed by an increase in the sensitivity of glucose transport to insulin. There is evidence suggesting that activation of p38 MAP kinase (p38) is involved in the stimulation of glucose transport by insulin and contractions. Exercise results in an increase in p38 phosphorylation that lasts for hours. In this context, we tested the hypothesis that activation of p38 results in an increase in insulin sensitivity. Muscles were exposed to anisomycin for 30 min to activate p38. Anisomycin increased p38 phosphorylation approximately 2.5-fold and glucose transport activity 2- to 3-fold. Three hours after anisomycin treatment, by which time the acute effect on glucose transport had partially worn off, sensitivity of muscle glucose transport to 60 microU/ml insulin was markedly increased. Both the activation of p38 and the increase in insulin sensitivity induced by anisomycin were completely prevented by pretreatment of muscles with the p38 inhibitor SB-202190. However, in contrast to the finding with anisomycin, inhibition of p38 activation did not prevent the contraction-induced increase in insulin sensitivity. Thus our results show that activation of p38 is followed by an increase in insulin sensitivity of muscle glucose transport. However, activation of p38 is not necessary for induction of an increase in muscle insulin sensitivity by contractions. This finding provides evidence that contractions have an additional effect that makes p38 activation unnecessary for enhancement of insulin sensitivity by contractile activity.     

Glucose transport by osmotic shock and vanadate is impaired by glucosamine

In 3T3-L1 adipocytes, we previously reported that glucosamine impairs insulin stimulation of glucose transport, which is accompanied by impaired insulin stimulation of serine/threonine kinase Akt. To examine the role of Akt in glucosamine-induced insulin resistance, we investigated time course for insulin stimulation of Akt activity and glucose transport during recovery from glucosamine-induced insulin resistance. After induction of insulin resistance by glucosamine, we washed cells to remove glucosamine and incubated them for various times. After one hour, insulin stimulated-glucose transport was significantly increased and continued to increase up to 6-24 h. Insulin stimulation of Akt, however, did not increase after 1-3 h and began to slightly increase after 6 h. Next, we investigated effects of osmotic shock and vanadate on glucose transport in glucosamine-treated cells and found that glucosamine completely inhibited their actions in these cells. These data suggest that an Akt-independent mechanism is operative in glucosamine-induced insulin resistance and glucosamine impairs glucose transport stimulated by various stimuli involving and not involving Akt activation.     

Mechanisms for the effects of acetylcholine on sodium transport in frog skin

Summary In frog skin (Rana temporaria) acetylcholine applied to the serosal surface produces either a sustained inhibition or sustained stimulation of short-circuit current (SCC). The former effect is accompanied by a reduction and the latter by an increase in total tissue conductance. Both effects of acetylcholine can be accounted for, within experimental error, by changes in net sodium flux across the tissue. By use of selective agonists and antagonists it is concluded that acetylcholine interacts with muscarinic receptors in the serosal membrane. The effects of cholinoceptor agents are also seen with isolated epithelium.The stimulatory effect of acetylcholine is potentiated by theophylline and blocked by inhibitors of prostaglandin synthetase and by mepacrine. It is suggested that acetylcholine stimulates transport by liberating prostaglandins which may then activate adenylcyclase. The inhibitory effect of acetylcholine is correlated with a reduction in cyclic AMP content of the epithelium. Calcium appears to be an important determinant of the type of response seen with acetylcholine, but the mechanism is not known.     

Insulin activates phospholipase C-gamma1 via a PI-3 kinase dependent mechanism in 3T3-L1 adipocytes

Previously we have shown that the insulin receptor and phospholipase C-gamma1 physically interact in the 3T3-L1 adipocyte cell line. In this study, we investigated the ability of insulin and PDGF to stimulate PLC-gamma1 enzyme activity as measured by PI-(4,5)P(2) hydrolysis. Both insulin and PDGF caused a rapid (<1 min) increase in PLC activity associated with the respective receptor. PDGF treatment resulted in a higher and more sustained stimulation of PLC-gamma1 activity compared to insulin (0.95 pmol/min/mg vs 0.68 pmol/min/mg). Furthermore, insulin and PDGF promoted increases in total cellular DAG, one of the products of PI-(4,5)P(2) hydrolysis. Insulin-stimulated PLC activity appears to be downstream of PI-3Kinase as the DAG increase was partially blocked by Wortmannin and addition of PI-(3,4,5)P(3) activated PLC-gamma1 in vitro. Inhibition of PLC using U73122 or an inhibitory peptide caused a decrease in insulin-stimulated 2-deoxyglucose transport and GLUT4 translocation that was rescued by the addition of OAG, a cell-permeable synthetic DAG.     

Insulin and epidermal growth factor. Human fibroblast receptors related to deoxyribonucleic acid synthesis and amino acid uptake.

Receptors for insulin and epidermal growth factor (EGF) have been studied in confluent cultured intact human fibroblast monolayers. 125-I-EGF binds specifically to fibroblast monolayers. Half-maximal binding is observed at 4 times 10 minus 10 M EGF; at saturation of binding approximately 4 times 10-4 molecules of EGF are bound per cell. 125-I-Insulin is also bound specifically by intact monolayers with half-maximal binding observed at 10 minus 9 M insulin; about 4 times 10-3 molecules of insulin are bound per cell at saturation. Both insulin and EGF stimulate thymidine incorporation and alpha-aminoisobutyrate uptake. A half-maximal effect for insulin is observed at about 10 minus 9 M, both for the stimulation of thymidine incorporation and for the stimulation of alpha-aminoisobutyrate uptake; for EGF, half-maximal stimulation of both thymidine incorporation and alpha-aminoisobutyrate uptake is observed at 10 minus 10 M EGF. EGF causes an apparent greater stimulation of thymidine incorporation than does insulin, whereas the stimulation of alpha-aminoisobutyrate uptake is the same for both insulin and EGF. The degree of stimulation of alpha-aminoisobutyrate uptake by either insulin or EGF varied (1.2- to 2-fold) from one batch of cells to another, as did the measured values of the apparent K-m (average value 1 mM, range 0.6 to 2 mM) and V-max (average, 0.82, range 0.78 to 0.87 nmol/100 mug of protein per min) for alpha-aminoisobutyrate. Nonetheless, the apparent K-m of each peptide for stimulation of alpha-aminoisobutyrate uptake was independent of the degree of increase in alpha-aminoisobutyrate uptake, and was constant from one batch of cells to another. The peptide-mediated stimulation of alpha-aminoisobutyrate uptake can be attributed to a decrease in the apparent K-m for alpha-aminoisobutyrate (e.g. for insulin) from 0.70 to 0.57 mM; for EGF from 0.87 to 0.66 mM) and a concomitant increase in the apparent V-max for alpha-aminoisobutyrate (e.g. for insulin from 0.78 to 0.87 and for EGF from 0.80 to 0.84 nmol/min/100 mug of cell protein). The stimulation requires a 40- to 60-min period of preincubation with either peptide and is blocked by pretreating cells with cycloheximide. In the presence of ouabain, both peptides inhibit rather than stimulate alpha-aminoisobutyrate uptake; ouabain lowers the basal rate of alpha-aminoisobutyrate uptake. The uptake of 3-0-methyl-D-glucose is not affected by either EGF or insulin under conditions where insulin stimulates fat cell transport. These observations indicate that cultured human fibroblasts possess specific binding sites for insulin and EGF, which sites can be related to two actions of the peptides: stimulation of thymidine incorporation and alpha-aminoisobutyrate uptake.     

Basolateral potassium membrane permeability of A6 cells and cell volume regulation

The K⁺ permeabilities (⁸⁶Rb(K) transport) of the basolateral membranes (J_bK) of a renal cell line (A6) were compared under isosmotic and hypo-osmotic conditions (serosal side) to identify the various components involved in cell volume regulation.Changing the serosal solution to a hypo-osmotic one (165 mOsm) induced a fast transient increase in Ca _i (max <1 min) and cell swelling (max at 3–5 min) followed by a regulatory volume decrease (5–30 min) and rise in the SCC (stabilization at 30 min). In isosmotic conditions (247 mOsm), the ⁸⁶Rb(K) transport and the SCC were partially blocked by Ba²⁺, quinidine, TEA and glibenclamide, the latter being the least effective. Changing the osmolarity from isosmotic to hypo-osmotic resulted in an immediate (within the first 3–6 min) stimulation of the ⁸⁶Rb(K) transport followed by a progressive decline to a stable value higher than that found in isosmotic conditions. A serosal Ca²⁺-free media or quinidine addition did not affect the initial osmotic stimulation of J_bK but prevented its secondary regulation, whereas TEA, glibenclamide and DIDS completely blocked the initial J_bK increase. Under hypo-osmotic conditions, the initial J_bK increase was enhanced by the presence of 1 mm of barium and delayed with higher concentrations (5 mm). In addition, cell volume regulation was fully blocked by quinidine, DIDS, NPPB and glibenclamide, while partly inhibited by TEA and calcium-free media.We propose that a TEA- and glibenclamide-sensitive but quinidine-insensitive increase in K⁺ permeability is involved in the very first phase of volume regulation of A6 cells submitted to hypo-osmotic media. In achieving cell volume regulation, it would play a complementary role to the quinidine-sensitive K⁺ permeability mediated by the observed calcium rise.This work was supported by grants from the Commissariat à l'Energie Atomique and the Centre National de Recherche Scientifique URA 638.     

Protein kinase Cdelta mediates insulin-induced glucose transport in primary cultures of rat skeletal muscle

Insulin activates certain protein kinase C (PKC) isoforms that are involved in insulin-induced glucose transport. In this study, we investigated the possibility that activation of PKCdelta by insulin participates in the mediation of insulin effects on glucose transport in skeletal muscle. Studies were performed on primary cultures of rat skeletal myotubes. The role of PKCdelta in insulin-induced glucose uptake was evaluated both by selective pharmacological blockade and by over-expression of wild-type and point-mutated inactive PKCdelta isoforms in skeletal myotubes. We found that insulin induces tyrosine phosphorylation and translocation of PKCdelta to the plasma membrane and increases the activity of this isoform. Insulin-induced effects on translocation and phosphorylation of PKCdelta were blocked by a low concentration of rottlerin, whereas the effects of insulin on other PKC isoforms were not. This selective blockade of PKCdelta by rottlerin also inhibited insulin-induced translocation of glucose transporter 4 (GLUT4), but not glucose transporter 3 (GLUT3), and significantly reduced the stimulation of glucose uptake by insulin. When overexpressed in skeletal muscle, PKCdelta and PKCdelta were both active. Overexpression of PKCdelta induced the translocation of GLUT4 to the plasma membrane and increased basal glucose uptake to levels attained by insulin. Moreover, insulin did not increase glucose uptake further in cells overexpressing PKCdelta. Overexpression of PKCdelta did not affect basal glucose uptake or GLUT4 location. Stimulation of glucose uptake by insulin in cells overexpressing PKCdelta was similar to that in untransfected cells. Transfection of skeletal myotubes with dominant negative mutant PKCdelta did not alter basal glucose uptake but blocked insulin-induced GLUT4 translocation and glucose transport. These results demonstrate that insulin activates PKCdelta and that activated PKCdelta is a major signaling molecule in insulin-induced glucose transport.     

Insulin stimulates transepithelial sodium transport by activation of a protein phosphatase that increases Na-K ATPase activity in endometrial epithelial cells.

The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na(+) transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na(+) transport two- to threefold by increasing Na(+)-K(+) ATPase transport activity and basolateral membrane K(+) conductance without increasing the apical membrane amiloride-sensitive Na(+) conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na(+) absorption with a further increase in Na(+)-K(+) ATPase transport activity and an increase in apical membrane amiloride-sensitive Na(+) conductance. The effect of insulin on the Na(+)-K(+) ATPase was the result of an increase in V(max) for extracellular K(+) and intracellular Na(+), and an increase in affinity of the pump for Na(+). Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of alpha-1 and alpha-2 isoforms, but not the alpha-3 isoform of Na(+)-K(+) ATPase, which did not change in the presence of insulin. Insulin-stimulated Na(+) transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)(3)], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na(+) transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3-kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na(+)-K(+) ATPase activation.     

Cellular lithium and transepithelial transport across toad urinary bladder

Summary Toad urinary bladders were exposed on either their mucosal or serosal surfaces, or on both surfaces, to medium in which sodium was replaced completely by lithium. With mucosal lithium Ringer's, serosal sodium Ringer's, short-circuit current (SCC) declined by about 50 percent over the first 60 min and was then maintained over a further 180 min. Cellular lithium content was comparable to the sodium transport pool. With lithium Ringer's serosa, SCC was abolished over 60 to 120 min whether the mucosal cation was sodium or lithium. Measurements of cellular ionic composition revealed that the epithelial cells gained lithium from both the mucosal and serosal media. With lithium Ringer's mucosa and serosa, cells lost potassium and gained lithium and a little chloride and water, but these changes in cellular ions could not account for the current flow across the tissue under these conditions, which must, therefore, have been carried by a transepithelial movement of lithium itself. The inhibition by serosal lithium of SCC was overcome by exposure of the mucosal surface of the bladders to amphotericin B. Thus it reflected, predominantly, an inhibition of lithium entry to the cells across the apical membrane. It is suggested that this inhibition is a consequence of cellular lithium accumulation.     

Targeting the signaling pathway of acylation stimulating protein

Acylation stimulating protein (ASP; C3adesArg) stimulates triglyceride synthesis (TGS) and glucose transport in preadipocytes/adipocytes through C5L2, a G-protein-coupled receptor. Here, ASP signaling is compared with insulin in 3T3-L1 cells. ASP stimulation is not Galpha(s) or Galpha(i) mediated (pertussis and cholera toxin insensitive), suggesting G(alphaq) as a candidate. Phospholipase C (PLC) is required, because the Ca(2+) chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester and the PLC inhibitor U73122 decreased ASP stimulation of TGS by 93.1% (P < 0.0.001) and 86.1% (P < 0.004), respectively. Wortmannin and LY294002 blocked ASP effect by 69% (P < 0.001) and 116.1% (P < 0.003), respectively, supporting phosphatidylinositol 3-kinase (PI3K) involvement. ASP induced rapid, transient Akt phosphorylation (maximal, 5 min; basal, 45 min), which was blocked by Akt inhibition, resembling treatment by insulin. Downstream of PI3K, mamalian target of rapaycin (mTOR) is required for insulin but not ASP action. By contrast, both ASP and insulin activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK(1/2)) pathway, with rapid, pronounced increases in ERK(1/2) phosphorylation, effects partially blocked by PD98059 (64.7% and 65.9% inhibition, respectively; P < 0.001). Time-dependent (maximal, 30 min) transient calcium-dependent phospholipase A(2) (cPLA(2))(-Ser505) phosphorylation (by MAPK/ERK(1/2)) was demonstrated by Western blot analysis. ASP signaling involves sequential activation of PI3K and PLC, with downstream activation of protein kinase C, Akt, MAPK/ERK(1/2), and cPLA(2), all of which leads to an effective and prolonged stimulation of TGS.     

The glucose transporter in human fibroblasts is phosphorylated in response to phorbol ester but not in response to growth factors

The possibility that the stimulation of hexose transport in human fibroblasts by phorbol myristate acetate (PMA), insulin, platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) is associated with phosphorylation of the glucose transporter has been investigated. The time and concentration dependencies of the stimulation of transport by these agents under conditions identical to those used for phosphorylation were determined. Each agent, when used at the concentration that resulted in the maximal increase in transport rate, elicited this effect within 30 min of exposure. The extent of stimulation ranged from 15 to 70%. For determination of phosphorylation of the glucose transporter, fibroblasts were incubated for 16 h with [32P]Pi and exposed to the agonist for 30 min; the transporter was then isolated from a detergent lysate of the cells by immunoprecipitation with a monoclonal antibody. Under these conditions, there was no phosphorylation of transporter in basal cells and only PMA caused detectable incorporation of phosphate into the transporter. Thus, it is unlikely that the stimulation of glucose transport by insulin, PDGF and EGF involve transporter phosphorylation.     

Insulin-stimulated alpha-(methyl)aminoisobutyric acid uptake in skeletal muscle. Evidence for a short-term activation of uptake independent of Na+ electrochemical gradient and protein synthesis.

1. The present study was designed to explore the mechanisms by which insulin stimulates system A of amino acid transport in extensor digitorum longus (EDL) muscles, by using a system A analogue, alpha-(methyl)aminoisobutyric acid (MeAIB). 2. Insulin stimulation of MeAIB uptake was noted after only 30 min of incubation and was maximal at 60 min. Kinetics of the insulin effect on MeAIB uptake were characterized by an increased Vmax. without modification of Km for MeAIB. 3. Incubation of EDL muscles with cycloheximide for 90 min did not modify MeAIB uptake in either the presence or the absence of insulin, indicating the independence of insulin action from protein synthesis de novo. Incubations for 180 min with cycloheximide caused a decrease in basal MeAIB uptake; however, the percentage stimulation of amino acid transport by insulin was unaltered. Basal MeAIB uptake was increased by incubation for 180 min, but under these conditions no change in the percentage effect of insulin was found. 4. Ouabain, gramicidin D, or both, markedly decreased basal MeAIB uptake by EDL muscle, but the percentage effect of insulin was unaltered. 5. We conclude that insulin action on amino acid transport through system A in muscle is rapid, is characterized by an increased Vmax., and is independent of protein synthesis de novo and the Na+ electrochemical gradient. Our data are compatible with insulin acting directly on the system A transporter.