Dependence of renal (Na+ + k+)-adenosine triphosphatase activity on thyroid status.

In thyroidectomized rats, a single injection of L-2,,5,2'-triiodothyronine (T3) (50mug/100 g body weight) elicited at 45% increase in (Na+ + k+)-dependent adenosine triphosphatase (NaK-ATPase) activity of the membrane-rich fraction of renal cortex at the optimal time of response, 48 h after injection. Three successive doses of T3 (50 mug/100 g body weight), given on alternate days, increased NaK-ATPase by 67% in the renal cortex but had no significant effect on the outer medulla or the papilla. Moreover, T3 had no effect on Mg2+-dependent adenosine trisphatase (MgATPase) in cortex, cedulla, or papilla. Three doses of T3 (50 mug/100 g body weight) given on alternate days to thyroidectomized rats elecited a 134, 79, and 46% increase in Vmax for ATP, Na4, and K+, respectively. There were no changes in the Km for ATP or the K1/2 values for Na+ and K+. Two methods were used to estimate the effect of T3 on the number of NaK-ATPase units (assumed to represent the number of Na+ pump sites); rat renal plasma membrane fractions were incubated with [gamma-32P]ATP, Mg2+, and Na+; the 32P-labeled membrane protein yeild was quantitatively dependent on Na+ and was hydrolyzed on addition of K+. There was a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of phosphorylated intermediate formed, in renal cortical membrane fractions from thyroidectomized rats given T3 or the diluent. There was also a linear correlation between the specific activity of NaK-ATPase (Vmax) and the amount of [3H]ouabain specifically bound (Na+-, Mg2+-, APT-dependent) to the NaK-ATPase preparation. Injection of T3 resulted in a 70% increase in NaK-ATPase activity, a 79% increase in formation of the phosphorylated intermediate, and a 65% increase in the [H]ouabain specifically bound to the NaK-ATPase system. The T3-dependent increases in Vmax for ATP, Na+, and K+ and the proportionate increases in the phosphorylated intermediate and in the amount of [3H]ouabain bound indicate that T3 increases the number of NaK-ATPase units and that this increase accounts for the increase in NaK-ATPase activity.     

Decreased thyroid hormone-stimulated oxygen consumption and glucose uptake in mononuclear blood cells from patients with autosomal dominant osteopetrosis type I

Nine patients, from four different families, with autosomal dominant osteopetrosis were investigated. They all had roentgenological type I disease, characterized by universal, symmetrical osteosclerosis and enlarged thickness of the cranial vault. All patients appeared clinically euthyroid. Thyroxine (T4) and tri-iodothyronine (T3) induced oxygen consumption and glucose uptake were studied in vitro in mononuclear blood cells from patients and control persons. Unstimulated oxygen consumption from patients and controls did not differ, and no difference in unstimulated glucose uptake was observed. The increase in T4 and T3 stimulated oxygen consumption was significantly lower in cells from patients with osteopetrosis (T4: 0.007 +/- 0.004 mumol/mg DNA per h, T3: 0.011 +/- 0.004 mumol/mg DNA per h) compared with controls (T4: 0.017 +/- 0.003 mumol/mg DNA per h, T3: 0.023 +/- -0.013 mumol/mg DNA per h; p less than 0.05, p less than 0.05). Cellular glucose uptake after T4 and T3 stimulation was significantly lower in patients (T4: 0.032 +/- 0.017 mmol/l per mg DNA per h, T3: 0.02 +/- 0.017 mmol/l per mg DNA per h) compared with controls (T4: 0.09 +/- 0.017 mmol/l per mg DNA per h, T3: 0.08 +/- 0.01 mmol/l per mg DNA per h; p less than 0.05, p less than 0.01). The reduced oxygen consumption and glucose uptake indicate thyroid hormone resistance which may be of pathogenetic importance for the development of autosomal dominant osteopetrosis type I.     

Stimulation of bumetanide-sensitive K+ transport in Swiss 3T3 fibroblasts by serum and mitogenic hormones

Rapidly growing Swiss 3T3 fibroblasts possess a bumetanide-sensitive K+ transport system that is dependent on both Na+ and Cl- ions; a smaller bumetanide-insensitive component of K+ transport is also present. In cells brought to the quiescent state by 8-11 days of incubation without a medium change, the bumetanide-sensitive rate of transport was reduced by 63%; the bumetanide-insensitive rate did not change. Removal of dialyzed fetal calf serum from the uptake medium resulted in a substantial reduction in bumetanide-sensitive uptake in both rapidly growing cells (33% reduction) and quiescent cells (68% reduction) but had no effect on bumetanide-insensitive uptake. Insulin was almost as effective as dialyzed fetal calf serum in stimulating bumetanide-sensitive uptake; insulin was maximally stimulatory at 2.5 micrograms/ml. The combination of insulin, epidermal growth factor, and arginine-vasopressin was maximally effective in stimulating both bumetanide-sensitive K+ uptake and 3H-thymidine incorporation in quiescent cells; bumetanide, however, did not interfere with the hormonal stimulation of DNA synthesis. Thus, the bumetanide-sensitive K+ transport system is not necessary for such stimulation to occur. Furthermore, concentrations of hormones which stimulated significant levels of DNA synthesis produced no elevation in the intracellular concentration of K+. We conclude that the bumetanide-sensitive pathway of K+ transport is modulated by serum and by mitogenic hormones, but does not play a role in the stimulation of DNA synthesis by these factors.     

Intracellular univalent cations and the regulation of the BALB/c-3T3 cell cycle

Addition of serum to density-arrested BALB/c-3T3 cells causes a rapid increase in uptake of Na+ and K+, followed 12 h later by the onset of DNA synthesis. We explored the role of intracellular univalent cation concentrations in the regulation of BALB/c-3T3 cell growth by serum growth factors. As cells grew to confluence, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) fell from 40 and 180 to 15 and 90 mmol/liter, respectively. Stimulation of growth of density-inhibited cells by the addition of serum growth factors increased [Na]i by 30% and [K+]i by 13-25% in early G0/G1, resulting in an increase in total univalent cation concentration. Addition of ouabain to stimulated cells resulted in a concentration-dependent steady decrease in [K+]i and increase in [Na+]i. Ouabain (100 microM) decreased [K+]i to approximately 60 mmol/liter by 12 h, and also prevented the serum- stimulated increase in 86Rb+ uptake. However, 100 microM ouabain did not inhibit DNA synthesis. A time-course experiment was done to determine the effect of 100 microM ouabain on [K+]i throughout G0/G1 and S phase. The addition of serum growth factors to density-inhibited cells stimulated equal rates of entry into the S phase in the presence or absence of 100 microM ouabain. However, in the presence of ouabain, there was a decrease in [K+]i. Therefore, an increase in [K+]i is not required for entry into S phase; serum growth factors do not regulate cell growth by altering [K+]i. The significance of increased total univalent cation concentration is discussed.     

3,5-T2 stimulates oxygen consumption, but not glucose uptake in human mononuclear blood cells.

L-thyroxine (T4), L-triiodothyronine (T3) and 3,5-di-iodothyronine (T2) rapidly (within 30 min) stimulated oxygen consumption in human mononuclear blood cells, whereas the D isomers of T4 and T3 and Triac had no stimulatory effect. Oxygen consumption was stimulated by the same magnitude by equimolar concentrations (5-500 nmol/l) of L-T4, L-T3 and 3,5-T2 reaching a plateau at 100 nmol/l of 0.025 umol/mg DNA x h. The stimulatory effects of T4 and T3, but not of T2 were inhibited by PTU. Glucose uptake was stimulated only by L-T4 and L-T3, whereas 3,5-T2, Triac and the D-isomers of T4 and T3 had no effect. The dose response curve reached an apparent maximum at 100 nmol/l of 0.30 mmol/l x mg DNA x h and PTU had no effect on iodothyronine stimulated glucose uptake. We conclude that 3,5-T2 is a significant intracellular stimulator of oxygen consumption, whereas T3 and T4 stimulate glucose uptake.     

Characterization of digitalis-like factors in human plasma. Interactions with NaK-ATPase and cross-reactivity with cardiac glycoside-specific antibodies

Much of the evidence for a physiologically important endogenous inhibitor of the sodium pump has been either contradictory or indirect. We have identified three discrete fractions in desalted deproteinized plasma from normal humans that resemble the digitalis glycosides in that they: are of low molecular weight; are resistant to acid and enzymatic proteolysis; inhibit NaK-ATPase activity; inhibit Na+ pump activity in human erythrocytes; displace [3H]ouabain bound to the enzyme; and cross-react with high-affinity polyclonal and monoclonal digoxin-specific antibodies but not with anti-ouabain or anti-digitoxin antibodies. An additional fraction cross-reacted with digoxin-specific antibodies but had no detectable activity against NaK-ATPase. The three inhibitory fractions differed from cardiac glycosides in that their concentration-effect curves in a NaK-ATPase inhibition and [3H]ouabain radioreceptor assays were steeper than unlabeled ouabain. This suggests that these inhibitors are not simple competitive ligands for binding to NaK-ATPase. In the presence of sodium, no fraction required ATP for binding to NaK-ATPase, and in the presence of potassium, only one fraction had the reduced affinity for the enzyme that is characteristic of cardiac glycosides. Unlike digitalis, all three NaK-ATPase inhibitory fractions stimulated the activity of skeletal muscle sarcoplasmic reticulum Ca-ATPase. The presence of at least three fractions in human plasma that inhibit NaK-ATPase and cross-react to a variable degree with different digoxin-specific antibody populations could explain much of the conflicting evidence for the existence of endogenous digitalis-like compounds in plasma.     

Glycolysis in quiescent cultures of 3T3 cells. Stimulation by serum, epidermal growth factor, and insulin in intact cells and persistence of the stimulation after cell homogenization.

Addition of serum to quiescent cultures of 3T3 cells rapidly increases lactic acid formation and subsequently stimulates cell division. The stimulation of lactic acid production is seen at high, saturating concentrations of extra-cellular glucose. It is dependent on the time of exposure and on the dose of serum and is not blocked by the addition of cycloheximide, puromycin, or actinomycin D. In contrast, serum only marginally affects glycolysis by rapidly growing 3T6 or SV40-3T3 cells. In addition to serum, epidermal growth factor (0.1 to 10 ng/ml) and insulin (10 to 500 ng/ml) cause a striking stimulation of glycolysis in quiescent 3T3 cells. Neither exogenous cyclic nucleotides nor ouabain effect the glycolytic response, but the presence of Ca2+ markedly influences the activation of glycolysis by epidermal growth factor and by insulin. A novel finding in this study is that homogenates prepared from quiescent cells treated with serum, epidermal growth factor, or insulin show increased glycolysis as compared with homogenates from nonstimulated cultures. This finding will allow further experimental analysis of the cause of increased glycolysis in rapidly proliferating cells.     

CD4-Negative cells bind human immunodeficiency virus type 1 and efficiently transfer virus to T cells

The ability of human immunodeficiency virus strain MN (HIV(MN)), a T-cell line-adapted strain of HIV, and X4 and R5 primary isolates to bind to various cell types was investigated. In general, HIV(MN) bound to cells at higher levels than did the primary isolates. Virus bound to both CD4-positive (CD4(+)) and CD4-negative (CD4(-)) cells, including neutrophils, Raji cells, tonsil mononuclear cells, erythrocytes, platelets, and peripheral blood mononuclear cells (PBMC), although virus bound at significantly higher levels to PBMC. However, there was no difference in the amount of HIV that bound to CD4-enriched or CD4-depleted PBMC. Virus bound to CD4(-) cells was up to 17 times more infectious for T cells in cocultures than was the same amount of cell-free virus. Virus bound to nucleated cells was significantly more infectious than virus bound to erythrocytes or platelets. The enhanced infection of T cells by virus bound to CD4(-) cells was not due to stimulatory signals provided by CD4(-) cells or infection of CD4(-) cells. However, anti-CD18 antibody substantially reduced the enhanced virus replication in T cells, suggesting that virus that bound to the surface of CD4(-) cells is efficiently passed to CD4(+) T cells during cell-cell adhesion. These studies show that HIV binds at relatively high levels to CD4(-) cells and, once bound, is highly infectious for T cells. This suggests that virus binding to the surface of CD4(-) cells is an important route for infection of T cells in vivo.     

Discrepancy between GLUT4 translocation and glucose uptake after ischemia

Objective: Low-flow ischemia results in glucose transporter translocation and in increased glucose uptake. After total ischemia in rat heart, we found no increase in glucose uptake. Here we test the hypothesis that total ischemia is associated with decreased activation of GLUT4 despite translocation. Methods: Isolated working hearts (n=70, Sprague–Dawley rats) were perfused for 70 min at physiological workload with Krebs–Henseleit buffer containing [2-³H]glucose (5 mmol/l, 0.05 μCi/ml) with either oleate (0.4 mmol/l, 1%BSA) or pyruvate (5 mmol/l, 1%BSA). After 20 min, hearts were subjected to 15 min of total ischemia followed by 35 min of reperfusion. We measured glucose uptake and intracellular free glucose (IFG) using [2-³H]glucose and [¹⁴C]sucrose, and determined the distribution of GLUT4 by colocalization immunofluorescence with Na–K ATP-ase. Results: Cardiac power was 10.1 ± 0.90 mW before ischemia and did not differ between groups. Recovery was the same in both groups (55.7 ± 24.8$%). Glucose uptake did not differ between groups before ischemia, and did not increase during reperfusion. Despite evidence of GLUT4 translocation after reperfusion in both groups, IFG did not increase compared with before ischemia. Conclusion: We conclude that there is a discrepancy between glucose transporter availability and glucose uptake after ischemia, which may be due to inhibition of GLUT4 in the plasma membrane. (Mol Cell Biochem 278: 129–137, 2005)     

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.     

Activators of AMP-activated protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1 adipocytes

To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P < 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin- and AMPK-activated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P < 0.05). AMPK- and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P < 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P < 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism.     

Endothelin stimulates glucose uptake and GLUT4 translocation via activation of endothelin ETA receptor in 3T3-L1 adipocytes

Endothelin-1 (ET-1) is a 21-amino acid peptide that binds to G-protein-coupled receptors to evoke biological responses. This report studies the effect of ET-1 on regulating glucose transport in 3T3-L1 adipocytes. ET-1, but not angiotensin II, stimulated glucose uptake in a dose-dependent manner with an EC50 value of 0.29 nM and a 2.47-fold stimulation at 100 nM. ET-1 stimulated glucose uptake in differentiated 3T3-L1 cells but had no effect in undifferentiated cells, although ET-1 stimulated phosphatidylinositol hydrolysis to a similar degree in both. The 3T3-L1 cells expressed approximately 560,000 sites/cell of ETA receptor, which was not altered during differentiation. Western blot analysis and immunofluorescence staining show that ET-1 stimulated the translocation of insulin-responsive aminopeptidase and GLUT4 to the plasma membrane. The effect of ET-1 on glucose uptake was blocked by A-216546, an antagonist selective for the ETA receptor. ET-1 treatment did not induce phosphorylation of insulin receptor beta-subunit, insulin receptor substrate-1, or Akt but stimulated the tyrosyl phosphorylation of a 75-kDa protein. Genistein (100 microM), an inhibitor of tyrosine kinases, inhibited ET-1-stimulated glucose uptake. Our results show that ET-1 stimulates GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes via activation of ETA receptor.     

Effect of thyroxine-binding globulin (TBG) on thyroxine (T4) uptake by human peripheral mononuclear cells: evidence of TBG-dependent uptake of T4

The effect of sialylated TBG and desialylated TBG on thyroxine (T4) uptake by human peripheral mononuclear cells was investigated in vitro. [125I]-T4 uptake was observed when the cells were incubated with free [125I]-T4. The uptake was inhibited in a concentration dependent manner when TBG was added. During the incubation, [125I]-T4 binding to TBG was observed. [125I]-T4 incorporation into cells was also observed when the cells were incubated with [125I]-T4-sialylated TBG or with [125I]-T4-desialylated TBG complex. The uptake was related to the temperature and length of time of the incubation. The amount of [125I]-T4 incorporated into the cells incubated with [125I]-T4-sialylated TBG was greater than that into the cells incubated with [125I]-T4-desialylated TBG during the early 0-20 min. incubation, whereas the amount of [125I]-T4 incorporated into the cells incubated with [125I]-T4-desialylated TBG became greater than that into the cells incubated with [125I]-T4-sialylated TBG after 20 min. of incubation. Pretreatment of the cells with methylamine blocked [125I]-T4 uptake in both cases, i.e. incubated with [125I]-T4-sialylated TBG and incubated with [125I]-T4-desialylated TBG. The results suggest that TBG plays a role not only as a carrier protein for T4 in circulation but also as a protein which can transport T4 from the extracellular into the intracellular space, so that the mechanism of T4 transport mediated by desialylated TBG is different from that mediated by sialylated TBG, and that the T4 transport system in both cases, mediated by sialylated TBG and by desialylated TBG, may be related to the internalization of T4-TBG-TBG receptor complex or of T4-T4 receptor complex if TBG receptors are present in the outer surface of the cell membrane.     

Diet and serum sex hormones in healthy men

The possible effect of dietary fat content and the ratio of polyunsaturated to saturated fatty acids (P/S-ratio) on serum sex hormones was studied in 30 healthy male volunteers. The customary diet of the subjects, which supplied 40% of energy as fat (mainly from animal sources, P/S-ratio 0.15) was replaced for a 6 weeks period by a practically isocaloric experimental diet containing significantly less fat (25% of energy) with a higher P/S-ratio (1.22) and other environmental factors were stabilized. Serum testosterone and 4-androstenedione decreased from 22.7 +/- 1.1 nmol/l to 19.3 +/- 1.2 nmol/l, (SEM, P less than 0.001) and from 4.6 +/- 0.2 nmol/l to 4.3 +/- 0.2 nmol/l (SEM, P less than 0.01), respectively. These changes were paralleled by a reduction in serum free (non-protein bound) testosterone (P less than 0.01) suggesting a possible change in biological activity. During the low fat period a significant negative correlation between serum prolactin and androgens was observed. All the changes in androgen levels were reversible. With the exception of a small but non-significant decrease in serum estradiol-17 beta, the other hormone parameters were practically unaffected by the dietary manipulation. Our results indicate that in men a decrease in dietary fat content and an increase in the degree of unsaturation of fatty acids reduces the serum concentrations of androstenedione, testosterone and free testosterone. The mechanism and importance of this phenomenon is discussed in the light of epidemiological and experimental data.     

Clinical evaluation of accuracy in determining serum free thyroxine and free triiodothyronine in patients with non-thyroidal illness: immunoglobulin effect on T3/TBG ratio and T4/TBG ratio

We examined the effect of endogenous immunoglobulins (G, A and M) and albumin on the measurement of thyroid hormones by different methods, including a new non-isotopic immunoassay of free thyroxine (FT4) and free triiodothyronine (FT3), in a large number of patients with non-thyroidal illness (NTI). Variations in serum protein concentrations can affect the results of radioimmunoassay of human thyroid hormones and thyroxine binding globulin (TBG). Our data revealed that in patients with non-thyroidal illness, when fluctuations in serum gamma-globulin occurred the T3/TBG and T4/TBG ratios altered. Consequently, when patients are suffering from non-thyroidal illness with changing gamma-globulin levels, clinical scientists should take care when they use T3/TBG and T4/TBG ratios as a substitute for FT3 or FT4 estimation. We found FT4 and FT3 (determined with Amerlex-M kits) T3 and the T3/TBG ratio were altered inversely due to the difference in the serum gamma-globulin levels. A recently developed enhanced luminescence enzyme immunoassay for FT3 and FT4 (Amerlite FT3 and FT4 kits) provides more reliable and accurate results, because of its resistance to interference, especially from albumin and gamma-globulin.     

Role of calcium in serum-stimulation of hexose transport in muscle cells

Serum stimulates glucose uptake into several cells in culture. In intact muscle, an increase in cytosolic free Ca2+ has been proposed to mediate the activation of glucose uptake by hormones and other stimuli [Cell Calcium (1980) 1, 311-325]. We report that hexose (2-deoxy-D-glucose) uptake into L6 muscle cells in culture is enhanced several-fold by fetal calf serum. The increase in uptake is due to stimulation of transmembrane transport, since serum also stimulated uptake of the non-metabolizable hexose 3-O-methyl-D-glucose. The role of Ca2+ in this stimulation was assessed: (i) stimulation of transport by serum was independent of the presence of extracellular Ca2+ during the incubation with serum; (ii) the intracellular levels of free Ca2+, measured by the fluorescence of the novel Ca-indicator quin-2, were identical in serum-stimulated and control cells. It is concluded that hexose transport can increase in muscle cells without concomitant changes in cytoplasmic free Ca2+.     

Nuclear thyroxine and triiodothyronine binding in mononuclear cells in dependence of age

Nuclear binding of thyroxine (T4) and triiodothyronine (T3) in mononuclear blood cells was investigated in 12 young (age 16-30 years) healthy subjects (group A), in 12 middle-aged (age 31-60 years) healthy subjects (group B) and in 12 elderly (61-90 years) healthy subjects. Serum free T3 was depressed in group C as compared to the younger age groups, whereas serum free T4 and TSH did not differ between the groups. Maximal specific nuclear binding capacity for both T4 and T3 decreased with increasing age, T4 group A: 1.2 fmol T4/100 micrograms DNA, group B: 1.2 fmol T4/100 micrograms DNA, group C: 0.7 fmol T4/100 micrograms DNA; T3 group A: 1.7 fmol T3/100 micrograms DNA, group B: 1.0 fmol T3/100 micrograms DNA, group C: 0.9 fmol T3/100 micrograms DNA. The equilibrium association constant (Ka) for T4 increased with age, group A: Ka = 3.3 X 10(9) l/mol, group B: Ka = 3.2 X 10(9) l/mol, group C: Ka = 6.4 X 10(9) l/mol, whereas Ka for nuclear binding of T3 decreased with age group A: Ka = 3.9 X 10(9) l/mol, group B: Ka = 5.9 X 10(9) l/mol, group C: Ka = 1.8 X 10(9) l/mol. We conclude that, whereas the opposite variations of nuclear capacity and binding affinity for T4 tend to preserve the nuclear T4 concentration, the nuclear T3 concentration definitely decreases with age. The unaltered serum levels of TSH suggest that the decrease of both serum levels of free T3 and the nuclear T3 concentration might represent physiologically changes in old age.     

Identification of NaK-ATPase inhibitors in human plasma as nonesterified fatty acids and lysophospholipids

Elevated plasma levels of factors with cardiac glycoside-like activity have been implicated in the response to volume expansion in animals and in the pathogenesis of certain human diseases. We recently described four fractions (IR1, EI1, EI2, EI3) from normal human plasma that inhibit NaK-ATPase, displace ouabain from the enzyme, and exhibit digoxin-like immunoreactivity (Kelly, R. A., O'Hara, D. S., Canessa, M. L., Mitch, W. E., and Smith, T. W. (1985) J. Biol. Chem. 260, 11396-11405). In this report, we identify the active component of these plasma fractions as long-chain nonesterified fatty acids (NEFA) and lysophospholipids. These lipids were present in fractions EI1, EI2, and EI3 in quantities sufficient to account for all of the NaK-ATPase inhibitory activity. The digoxin-like immunoreactivity in fraction IR1 could be attributed to hydrocortisone and other endogenous steroids. To explore the nature of the lipid-NaK-ATPase interactions, we examined the effects of various ATP or sodium concentrations on the NaK-ATPase activity measured in the presence of NEFA. Varying sodium did not affect the inhibition of NaK-ATPase by linoleic acid. At less than 0.15 mM ATP, linoleic acid stimulated NaK-ATPase, but at higher ATP concentrations, the enzyme was progressively inhibited. In summary, NEFA and lysophospholipids, at levels similar to those occurring in human plasma, may account for all of the NaK-ATPase inhibitory activity observed in human plasma fractions. These lipids probably do not directly regulate NaK-ATPase in vivo under normal physiologic conditions, but may alter the sodium pump in disease states characterized by abnormalities in lipid metabolism or plasma protein binding.     

Efficacy of glucose, ouabain and an aldose reductase inhibitor on 2-[3H] myo-inositol uptake by human, rat and rabbit erythrocytes

Myo-inositol uptake by erythrocytes from humans, rabbits and rats was studied with an isotope technique. In human erythrocytes, the inhibitory effect on myo-inositol uptake was stronger with glucose than with ouabain. However, an aldose reductase inhibitor (ONO-2235, 100 microM) or insulin (200 microU/ml) failed to correct the decrease in myo-inositol uptake in packed RBC, produced by either 10 mM glucose or 2mM ouabain. Ten mM ouabain had an inhibitory effect on myo-inositol uptake in all species, but an inhibitory effect was not observed with 20 mM glucose in rabbit erythrocytes. The results suggest that myo-inositol uptake by erythrocytes may be dependent on the active transport system via sodium-ATPase and that erythrocytes may not be a suitable model to monitor the possible effect of an aldose reductase inhibitor on myo-inositol concentrations in other tissues concerned with diabetic complications.     

Differential regulation of NHE1 phosphorylation and glucose uptake by inhibitors of the ERK pathway and p90RSK in 3T3-L1 adipocytes

Insulin stimulates trafficking of GLUT4 to the cell surface for glucose uptake into target cells, and phosphorylation of Ser703 of the Na⁺/H⁺ exchanger NHE1, which activates proton efflux. The latter has been proposed to facilitate optimal glucose uptake into cardiomyocytes. We found that the insulin-stimulated phosphorylation of Ser703 of NHE1 is mediated by p90RSK but not directly coupled to glucose uptake in 3T3-L1 adipocytes in the short-term. Inhibiting Erk1/2 activation prevented NHE1 phosphorylation but not glucose uptake in 3T3-L1 adipocytes. In contrast, both NHE1 phosphorylation and insulin-stimulated uptake of glucose into 3T3-L1 adipocytes were blocked by inhibitors of the N-terminal kinase domain of p90RSK, namely BI-D1870 and SL0101, but not the FMK inhibitor of the C-terminal kinase domain of p90RSK, though in our hands FMK did not inhibit p90RSK in 3T3-L1 adipocytes. Further experiments were consistent with phosphorylation of AS160 by PKB/Akt mediating insulin-stimulated trafficking of GLUT4 to the plasma membrane. BI-D1870 and SL0101 however, inhibited glucose uptake without blocking GLUT4 translocation. While BI-D1870 partially inhibited insulin-stimulated PKB activation in these cells, this only partially inhibited AS160 phosphorylation and did not block GLUT4 trafficking, suggesting that p90RSK might regulate glucose transport after GLUT4 translocation. Moreover, BI-D1870 also prevented PMA-induced glucose transport in 3T3-L1 adipocytes further suggesting a role for p90RSK in regulating uptake of glucose into the cells. Kinetic experiments are consistent with SL0101 being a direct competitor of 2-deoxyglucose entry into cells, and this compound might also inhibit uptake of glucose into cells via inhibiting p90RSK, as revealed by comparison with the inactive form of the inhibitor. Taken together, we propose that BI-D1870 and SL0101 might exert their inhibitory effects on glucose uptake in 3T3-L1 adipocytes at least partially through a p90RSK dependent step after GLUT4 becomes associated with the plasma membrane.