Uptake of thyroid hormone by isolated rat liver cells

L-Triiodothyronine is taken up by isolated rat liver cells by a process which is saturable and exhibits sigmoidity. Two uptake systems make themselves evident: A system with high affinity with an apparent K_t value of 52±22 nM and a system with low affinity with an apparent K_t value of 1446±764 nM. Cells heated at 60°C or after freezing do not show saturability of uptake. KCN inhibits the uptake by the low affinity system. In the presence of L-thyroxine and L-tyrosine the uptake of L-triiodothyronine is increased. The results suggest transport of L-triiodothyronine by proteins in the plasma membrane of the liver cell.     

Uptake and metabolism of N-(4'-pyridoxyl)amines by isolated rat liver cells.

Uptake and metabolism of [3H]pyridoxine and 3H-labeled N-(4'-pyridoxyl)amines by isolated rat liver cells were studied at physiological concentration (0.5 microM) of vitamin B6 by using both membrane filtration and centrifugation methods for removal of radiolabeled solutes after incubations with cells. It was found that the characteristics of import of N-(4'-pyridoxyl)amines into liver cells is similar to those of import of natural vitamin B6. Upon entry each 4'(N)-substituted pyridoxamine was converted to its 5'-phosphate and then oxidized to release pyridoxal 5'-phosphate and the original amine. Considerable size of the amine substituent is tolerated for transport and metabolism, but a charged function impedes entry. The amount of released pyridoxal 5'-phosphate (and therefore the amount of released original amine) is controlled partially by the size of the amine affixed to B6 and partially by the enzymatic steps involved. This system illustrates how biologically active amines can be piggybacked onto a vitamin that gains facilitated entry to cells that have the enzymatic means to release the free amine for subsequent effects within the cell.     

Insulin-like effects of histones H3 and H4 on isolated rat adipocytes

Crude preparations of histones had insulin-like actions in isolated adipocytes. This activity was attributed to the arginine-rich histones, H3 and H4. The metabolic effects of purified H3 and H4 on isolated adipocytes were similar to those of insulin in a number of respects. Like insulin, H3 and H4 stimulated the incorporation of both glucose and pyruvate in isolated cells and stimulated intercellular oxidation of glucose; in contrast, the lipolytic agents ACTH and isoproterenol actually inhibited the incorporation of pyruvate into adipocytes. In contrast to the effects of the lipolytic hormones, the effects of H3 and H4, like insulin, were not blocked by the presence of adenosine deaminase in the medium. The same concentrations of phenylarsine oxide were required to inhibit the stimulation of glucose incorporation whether by insulin or by histones. Furthermore, the addition of H4 or insulin to isolated adipocytes resulted in the increased phosphorylation of 17 kDa phosphoproteins as detected by two-dimensional electrophoresis. The insulin-like effect of the active histones was specific to their structure. Lysine-rich histones (H1, H2A and H2B), various polycations, and proteolytic fragments of purified H3 or H4 were all inactive. It is unknown whether this phenomenon might imply a physiological function for such endogenous molecules; however, a comparison of the detailed effects of insulin and histones might be informative in terms of common intracellular transduction systems.     

Uptake studies in adipocytes isolated from rainbow trout (Salmo gairdnerii). A comparison with adipocytes from rat and cat

Adipocytes were isolated from mesenteric adipose tissue of rainbow trout (Salmo gairdnerii) by incubation of tissue slices at 20 degrees C in a buffer containing 3 mg collagenase per ml. These cells were compared to adipocytes from the cat and the rat, isolated by conventional technique (1 mg collagenase per ml buffer, incubation temperature 37 degrees C). Uptake studies of some metabolites were performed with fish, rat and in some cases cat adipocytes. At a glucose concentration of 0.33 mM, the glucose uptake into rat cells was more than twice as fast as in cells from the cat, and more than five times as fast as in trout cells. 2-Amino butyrate resembled glucose in relative uptake rates between species. Metabolite uptake into rat cells was specific, with different uptake rates for different metabolites. The uptake into trout adipocytes proceeded at similar rates for all metabolites tested, provided the concentrations were the same. The uptake rate of glucose into rat cells was stimulated by insulin. Insulin had no effect on glucose uptake into adipocytes from trout.     

Fluid-phase endocytosis by isolated rat adipocytes

We have developed an assay, which uses radiolabeled sucrose as the marker, to measure the rate of fluid-phase endocytosis in isolated rat adipocytes. In addition, the assay was adapted to allow measurement of the release of sucrose from previously loaded cells (fluid-phase exocytosis). Adipocytes take up sucrose at an approximately linear rate for at least 1.5 hours. A portion of the pinocytosed sucrose is rapidly (half-time about 20 minutes) returned to the medium. The minimal value for fluid uptake by endocytosis is 57 nl/10(6) cells-h at 37 degrees C; this value corresponds to the formation of 110,000 endocytic vesicles of 100-nm diameter per cell per hour and the internalization of about 20% of the plasma membrane per hour. Insulin caused a small and variable increase in the rate of sucrose uptake. The average increase of 31% from 11 experiments is statistically significant at the level of P less than 0.01. A small insulin effect upon the uptake of the calcium complex of [14C]EDTA was also observed. Since this complex was taken up at 2.5 times the rate of sucrose, it probably entered by a combination of fluid-phase and adsorptive pinocytosis. Insulin did not elicit a significant change in the rate of sucrose release from preloaded cells.     

Dehydroascorbic acid transport by GLUT4 in Xenopus oocytes and isolated rat adipocytes

Dehydroascorbic acid (DHA), the first stable oxidation product of vitamin C, was transported by GLUT1 and GLUT3 in Xenopus laevis oocytes with transport rates similar to that of 2-deoxyglucose (2-DG), but due to inherent difficulties with GLUT4 expression in oocytes it was uncertain whether GLUT4 transported DHA (Rumsey, S. C. , Kwon, O., Xu, G. W., Burant, C. F., Simpson, I., and Levine, M. (1997) J. Biol. Chem. 272, 18982-18989). We therefore studied DHA and 2-DG transport in rat adipocytes, which express GLUT4. Without insulin, rat adipocytes transported 2-DG 2-3-fold faster than DHA. Preincubation with insulin (0.67 micrometer) increased transport of each substrate similarly: 7-10-fold for 2-DG and 6-8-fold for DHA. Because intracellular reduction of DHA in adipocytes was complete before and after insulin stimulation, increased transport of DHA was not explained by increased internal reduction of DHA to ascorbate. To determine apparent transport kinetics of GLUT4 for DHA, GLUT4 expression in Xenopus oocytes was reexamined. Preincubation of oocytes for >4 h with insulin (1 micrometer) augmented GLUT4 transport of 2-DG and DHA by up to 5-fold. Transport of both substrates was inhibited by cytochalasin B and displayed saturable kinetics. GLUT4 had a higher apparent transport affinity (K(m) of 0.98 versus 5.2 mm) and lower maximal transport rate (V(max) of 66 versus 880 pmol/oocyte/10 min) for DHA compared with 2-DG. The lower transport rate for DHA could not be explained by binding differences at the outer membrane face, as shown by inhibition with ethylidene glucose, or by transporter trans-activation and therefore was probably due to substrate-specific differences in transporter/substrate translocation or release. These novel data indicate that the insulin-sensitive transporter GLUT4 transports DHA in both rat adipocytes and Xenopus oocytes. Alterations of this mechanism in diabetes could have clinical implications for ascorbate utilization.     

Mechanisms of insulin degradation by isolated rat adipocytes

Summary We have examined some of the chemical and biological characteristics of the insulin-derived cell-associated radioactivity following incubation of isolated adipocytes with ¹²⁵I-insulin (10^–10 M) for one hour at 37 °C S ephadex G-50 chromatography of the cell-associated radioactivity demonstrated three peaks: peak I eluted with the void volume and consisted of large molecular weight material; peak II comigrated with 1251-insulin; and peak III consisted of small molecular weight degradation products (probably iodotyrosine). When the insulin peak (peak II) was divided into fourths, it was found that the binding and biologic activity of this material was not homogenous; thus, binding and biologic activity (relative to native insulin) fell markedly from the earliest to the latest eluting fractions of this peak. Furthermore, when the entire peak 11 material was applied to DEAE-Sephacel and eluted with a 0.01–0.2 M NaCl gradient, three distinct peaks were observed. These peaks were all 90% TCA precipitable, whereas the ability of the latter two eluting peaks to precipitate with anti-insulin antiserum was markedly reduced. When similar experiments were performed with chloroquine-treated cells, a large increase in cell-associated radioactivity was observed, and Sephadex G-50 chromatography demonstrated that this increase was entirely confined to peaks I and II. When the insulin peak (peak II) was divided into fourths, it was found that chloroquine markedly inhibited the decreased binding and biologic activity, from the earliest to the latest eluting fraction of this peak. Furthermore, when the peak II material (Sephadex G-50) from chloroquine-treated cells was chromatographed on DEAE-Sephacel, this material eluted in a single peak which was 95% TCA precipitable and 106% precipitable by anti-insulin antiserum. In conclusion, these studies demonstrate that: 1) intermediate insulin-derived products with reduced binding and biologic activity are generated in the process of cellular insulin degradation, and 2) the formation of these intermediate products is mediated by a chloroquine-sensitive pathway.     

Effects of thyroid hormones and reverse-triiodothyronine (rT3) pretreatment on beta-adrenoreceptors in the rat heart

Effects of thyroxine (T4), triiodothyronine (T3) or reverse-triiodothyronine (rT3) in vivo pretreatment or the effect of hypothyroidism on properties of beta-adrenoreceptors in the rat heart were investigated. beta-adrenergic antagonist hydroxybenzylpindolol (HYP125I) was used to estimate the maximal binding capacity (MBC) and the affinity (KD) of beta-adrenoreceptors. Both T4 and T3 in dose and time dependent manner increased MBC value but only slightly and insignificantly affected affinity of the beta-adrenoreceptor. The effect of T3 on MBC was higher than that of an equal dose of T4, but the latter effect persisted longer than that of T3. Hypothyroidism decreased MBC value, but increased the affinity of beta-adrenoreceptors. Pretreatment of rats with rT3 produced changes in beta-adrenoreceptors similar to those seen in hypothyroid rats.     

Uptake and 25-hydroxylation of vitamin D3 by isolated rat liver cells

The physiological roles played by hepatocytes and nonparenchymal cells of rat liver in the metabolism of vitamin D3 have been investigated. Tritium-labeled vitamin D3 dissolved in ethanol was administered intravenously to two rats. Isolation of the liver cells 30 and 70 min after the injection showed that vitamin D3 had been taken up both by the hepatocytes and by the nonparenchymal liver cells. The relative proportion of vitamin D3 that accumulated in the nonparenchymal cells increased with time. Perfusion of the isolated rat liver with [3H] vitamin D3 added to the perfusate confirmed the ability of both cell types to efficiently take up vitamin D3 from the circulation. By a method based on high pressure liquid chromatography and isotope dilution-mass fragmentography it was found that isolated liver cells in suspension had a considerable capacity to take up vitamin D3 from the medium. About 2.5 fmol of vitamin D3 were found to be associated with each hepatocyte or nonparenchymal cell after 1 h of incubation. 25-Hydroxylation in vitro was found to be carried out only by the hepatocytes. The rate of hydroxylation was about the same whether the cells were isolated from normal or rachitic rats (3.5 and 4 pmol of 25-hydroxyvitamin D3 formed per h per 10(6) cells, respectively). The possibility that the nonparenchymal cells might serve as a storage site for vitamin D3 in the liver is discussed.     

Uptake and metabolism of S-adenosyl-L-methionine by isolated rat hepatocytes

In the present study, direct evidence is given to SAMe capability of crossing the membrane of isolated rat hepatocytes. The kinetics of SAMe uptake is biphasic: a fast phase being completed in less than 15 sec and a slower one with an apparent K_m of 8.33 μM and a V_max of 10.6 pmol/min/mg protein. Both processes are pH and temperature dependent. Analysis of the fast phase by a Scatchard plot discloses two sets of binding sites of high and low affinity, respectively. Experiments carried out incubating isolated hepatocytes with double-labelled SAMe (methyl-³H, carboxyl-¹⁴C) have shown that about 70% of SAMe uptake by the cell is rapidly decarboxylated.     

Uptake of thiamin by isolated rat liver mitochondria

Investigation is made here of 14C-thiamin uptake by rat liver mitochondria in vitro. Following incubation with mitochondria, 14C-thiamin remained in the mitochondrial pellet in spite of several washings of the organelles. Accordingly, externally added thiamin produced intra/extra-mitochondrial concentration ratios up to 5.4 and 14C-thiamin space/3H2O space ratios higher than one. These ratios decreased with increasing vitamin concentrations, thus suggesting the occurrence of saturation characteristics for vitamin uptake into mitochondria. Thiamin was proven to enter both intermembrane and matrix spaces, where neither binding to intramitochondrial protein nor phosphorylation were found to occur. Moreover thiamin uptake inhibition by both metal ions and certain thiamin analogues was also found.     

Effect of thyroid hormones on 5'-nucleotidase of isolated rat fat cells.

5'-Nucleotidase was measured in isolated fat cells from normal, hypothyroid and hyperthyroid rats. This was done to find out whether thyroid hormones had an effect on the production of adenosine by the fat cell. The results showed that 5'-nucleotidase is modified when the rats received injections of 3,3',5-triiodo-L-thyronine (T3). There was no change in the enzyme in hypothyroidism or when T3 was added to incubation of cells.     

Phosphatidylinositol-3-kinase in isolated rat adipocytes. Activation by insulin and subcellular distribution.

Insulin increases phosphatidylinositol-3-kinase (PI-3-kinase) activity in Chinese hamster ovary cells transfected with human insulin receptor (Ruderman, N. B., Kapeller, R., White, M. F., and Cantley, L. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415). The subcellular distribution of PI-3-kinase has not been investigated, and it is unclear if insulin has a stimulatory effect on PI-3-kinase in a nonproliferating target tissue, and, if so, whether this effect is subject to counter-regulation. To address these questions, we studied the effect of insulin on PI-3-kinase activity in isolated rat adipocytes. Activity was measured in plasma membranes, intracellular membranes, and cytosol of control and insulin-treated adipocytes, and in anti-Tyr(P) immunoprecipitates prepared from these fractions and from whole cell lysates. Treatment of adipocytes with insulin (200 nM) caused a half-maximal increase in anti-Tyr(P)-immunoprecipitable PI-3-kinase activity in whole cell lysates within 2 min. This effect was concentration-dependent, and it was sensitive to inhibition by norepinephrine. In insulin-stimulated cells, 75% of anti-Tyr(P)-immunoprecipitable PI-3-kinase activity was found in the low density microsomes. This fraction also exhibited the highest specific activity of PI-3-kinase, and insulin caused a further increase in this activity. Anti-Tyr(P)-immunoprecipitable PI-3-kinase activity was also found in the plasma membranes of insulin-treated cells, but this accounted for only a minor portion of the total and anti-Tyr(P)-immunoprecipitable PI-3-kinase activity. The majority of PI-3-kinase activity (90%) in control cells was cytosolic, but this was not increased in response to insulin nor was it anti-Tyr(P)-immunoprecipitable. These data demonstrate that insulin increases the activity of PI-3-kinase in adipocytes and this effect is subject to inhibition by a physiological antagonist of insulin action. The data also indicate that the effect of insulin to increase PI-3-kinase activity is expressed primarily in the low density intracellular membranes and to a lesser extent in the plasma membranes.     

Uptake and degradation of asialo-fetuin by isolated rat hepatocytes.

125I-labelled asialo-fetuin was taken up by isolated rat hepatocytes by a saturable process. Half maximum uptake was seen at about 3 . 10(-8) M asialo-fetuin. Rate of uptake of asialo-fetuin exceeded rate of degradation at all concentrations of asialo-fetuin tested. Degradation of asialo-fetuin, as indicated by release of acid-soluble radioactivity from the cells, was inhibited by NH4Cl and chloroquine. The intracellular distribution of labelled asialo-fetuin was studied by differential and density gradient centrifuging. The distribution curves for radioactivity indicated that asialo-fetuin was present in lysosomes about 1 h after the uptake had started. Chloroquine and ammonium ions seemed to inhibit the uptake of asialo-fetuin into the lysosomes, possibly by interfering with the fusion between phagosomes and lysosomes.     

Glucose tolerance factor stimulates 3-O-methylglucose transport into isolated rat adipocytes

Glucose tolerance factor partially purified from yeast extract powder stimulated [U-14C]-D-glucose uptake to a level 5.6 times greater than the basal level in the absence of insulin in isolated adipocytes prepared from rats fed with normal laboratory chow. The factor also stimulated 3-O-methylglucose transport 2.2-fold from the basal level in the absence of insulin, but not in the presence of 8 nM insulin. Kinetic analysis revealed that glucose tolerance factor increased 3-O-methylglucose transport by decreasing the Ks value for 3-O-methylglucose with little change in the Vmax.