Regulation of the d-glucose transport system in isolated fat cells

Summary Recent technical advances have yielded considerable new biochemical insights into the hexose transport systems of both brown and white fat cells. In the present studies a novel filtration method was used to monitor initial rates of 3-O-(³H) methylglucose uptake in isolated white fat cells. Transport of 3-O-methylglucose, a non-metabolizable analogue of glucose, occurred by facilitated diffusion, was inhibited by glucose, phloridzin, cytochalasin B and dipyridamole, and was rapidly stimulated by insulin as well as lectins. Total 3-O-methylglucose uptake in white fat cells could be attributed to two kinetically distinct processes in addition to a certain degree of diffusion.Two important new features of glucose transport in fat cells have been discovered. First, in both brown and white fat cells transport per se does not appear to be necessarily rate-limiting for further glucose metabolism. Thus vitamin K₅, which markedly increases glucose oxidation by brown fat cells, did not affect the glucose transport system activity. Glucose utilization can apparently be significantly enhanced in fat cells by agents which either increase transport system activity or intracellular enzyme activity. Second, the transport system itself, whether in the basal state or after activation by insulin, lectins, or oxidants, is resistant to sulfhydryl reagents such as N-ethylmaleimide, while the increase in transport activity due to these agents is exquisitely sensitive to sulfhydryl blockage. N-ethylmaleimide blocks the stimulatory effect of insulin on transport whereas addition of insulin to fat cells prior to the reagent completely protects against this inhibitory effect. Further, N-ethylmaleimide prevents the elevated rates of transport system activity due to insulin (or other agents) from returning to basal levels once the cells are washed free of hormone. These data are consistent with the concept that activation of the transport system involves oxidation of key membrane sulfhydryls to the disulfide form, but alternative models are also possible. In any case, these findings provide a possible biochemical clue for future studies designed to identify the specific component(s) involved in the regulatory mechanism which modulates transport of glucose in isolated fat cells.Invited ArticleRecipient of the Elliot P. Joslin Research and Development Award of the American Diabetes Association.     

Regulation of the ColV plasmid-determined iron (III)-aerobactin transport system in Escherichia coli.

Regulation by iron was studied in Escherichia coli strains whose iron supply was entirely dependent on the iron(III)-aerobactin system determined by the ColV plasmid. By the insertion of phage Mu (Ap lac) into the ColV plasmid, mutants were selected that could no longer grow in iron-limited media. The inserted Mu (Ap lac) strongly reduced the amount of aerobactin and he cloacin receptor protein formed by the cells. Their production was no longer subject to regulation by iron. The Mu (Ap lac) insertion apparently led to a polar effect on the expression of the presumably closely linked genes that control the synthesis of aerobactin and the cloacin receptor protein. The expression of the beta-galactosidase gene on the inserted phage genome came under the control of the iron state of the cells. Under iron-limited growth conditions, the amount of beta-galactosidase synthesized was, depending on the strain studied, 6 to 30 times higher than under iron-sufficient growth conditions. In fur mutants with an impaired iron regulation of ll iron supply systems studied so far, high amounts of beta-galactosidase were synthesized independent of the cells' iron supply. The results demonstrate an iron-controlled promoter on the ColV plasmid which is subject to regulation by the chromosomal fur gene.     

Regulation of the adenylyl cyclase signaling system in various types of cultured endothelial cells

We studied the effects of modulators of the adenylyl cyclase pathway on the accumulation of cAMP in endothelial cells isolated from bovine aortas, pig pulmonary arteries, human umbilical veins, and human subcutaneous adipose microvessels. In addition to quantitative differences in the basal levels, cAMP stimulation in different endothelial cell types varied in sensitivity and magnitude in response to both the direct adenylyl cyclase activator forskolin and the β-adrenergic receptor agonist isoproterenol. Furthermore, the ubiquitous phosphodiesterase inhibitor IBMX differentially enhanced both the basal and the stimulated cAMP levels in the various cell types. Histamine caused an elevation of cAMP only in bovine aortic endothelial cells and in human umbilical vein endothelial cells. Treatment of the cells with cholera and pertussis toxins, which uniquely affect G-protein subunits, resulted in divergent elevation of cAMP in the various cells. Thus, in each cell type, a distinct profile of regulation of the cAMP levels was found. Our results suggest that the adenylyl cyclase signaling system in various types of endothelial cells can be differentially regulated at the levels of receptors, G-proteins, adenylyl cyclase, and phosphodiesterase.     

Regulation of Na,K-pump-mediated transport by prolactin in cultured human prostate epithelial cells.

The prostate gland is unique in its ability to secrete large amounts of zinc and citrate, suggesting that it employs unusual transport mechanisms. Intracellular ionic homeostasis in prostate is likely to be mediated by the Na,K-pump, yet there have been few studies of its regulation in this tissue. Accordingly, we explored the expression of the Na,K-pump in PC3 cells, an established cell line of human prostate epithelial cells. Total RNA from confluent monolayers of PC3 cells was isolated, reverse transcribed, and the resulting complementary DNA was amplified by polymerase chain reaction using primers specific for each of the pump's constituent subunits. The amplification revealed a complex pattern of Na,K-pump expression, with detection of mRNAs encoding the alpha1-, alpha3-, alpha4-, betal-, beta2- and beta3-isoforms. We next examined the effect on pump activity of prolactin, an important mediator of cell proliferation in prostate cancer. Monolayers exposed to 10 nM prolactin for 24 hr revealed an inhibition of 40% in ouabain-sensitive 86Rb+ uptake, a sensitive measure of pump-mediated transport. These experiments suggest that the unique transport properties of prostate may depend, at least in part, on a complicated pattern of Na,K-pump expression and regulation.     

Regulation of glucose transport by angiotensin II and glucose in cultured vascular smooth muscle cells

Glucose transport in response to angiotensin II (AII) was assessed in cultured vascular smooth muscle (VSM) cells by measuring the uptake of [³H]-2-deoxyglucose, a radiolabeled non-metabolizable glucose analog. Significant stimulation occurred by 2 hr of exposure with the maximum effect being observed between 6 and 8 hr. AII effects were concentration dependent with a threshold response being detected at 0.1 nM. AII-stimulated transport was blocked by saralasin, an AII receptor antagonist, indicating that AII binding to a specific receptor is required for AII to elicit the transport response. AII-stimulated transport was also blocked when cells were incubated with cycloheximide for 6 hr, suggesting that protein synthesis is required for the long-term effects of AII on glucose transport. A specific protein synthesized in response to AII stimulation was the GLUT 1 glucose transporter as assessed by western blot analysis. Inhibition of protein kinase C (PKC) by bisindolylmaleimide and staurosporine did not affect VSM responsiveness to AII, suggesting that AII is capable of stimulating glucose transport through a PKC-independent mechanism; however, VSM responsiveness to AII did appear to be dependent upon the presence of extracellular calcium. The importance of calmodulin in mediating the response of VSM cells to AII was indicated by the inhibition of AII-stimulated glucose transport when VSM cells were incubated in the presence of the calmodulin inhibitors, calmidazolium and W7. Finally, glucose uptake increased with decreasing levels of glucose in the incubation medium. This was accompanied by a corresponding decrease in the relative effectiveness of AII in stimulating glucose uptake. J. Cell. Physiol. 177:94–102, 1998. © 1998 Wiley-Liss, Inc.     

Sulfate transport in cultured tobacco cells

Sulfate transport by tobacco (Nicotiana tabacum L. var. Xanthi) cells cultured on either l-cysteine or sulfate as a sole sulfur source was measured. The transport rate on either sulfur source was low during pre-exponential growth, increased during exponential growth, and was maximal in late exponential cells. The initial increase in transport rate was correlated with a decline in the intracellular sulfate, but was not correlated with the amino acid content of the cells which remained relatively constant before the depletion of the endogenous sulfate pool. The previously reported inhibition of sulfate transport by l-cysteine was shown to be caused by an elevation in intracellular sulfate resulting from the degradation of cysteine to sulfate. It is proposed that the intracellular sulfate pool is the major factor regulating the entry of sulfate into tobacco cells.     

Thymidine transport in cultured mammalian cells. Kinetic analysis,temperature dependence and specificity of the transport system

The transport of thymidine has been characterized kinetically and thermodynamically in Novikoff rat hepatoma cells grown in culture and, less extensively, in mouse L cells, Chinese hamster ovary cells, P388 murine leukemia cells and HeLa cells. That the characterizations pertained to the transport system per se was ensured, (i) by employing recently developed methods for rapid sampling of cell/substrate mixtures in order to follow isotope movements within a few seconds after initial exposure of cells to substrate; (ii) by utilizing cells rendered, by genetic or chemical means, incapable of metabolizing thymidine; and, (iii) by demonstrating conformity of the transport data to an integrated rate equation derived for a simple, carrier-mediated system. The results indicate that thymidine is transported into mammalian cells by a functionally symmetrical, non-concentrative system for which the carrier : substrate dissociation constant ranges from about 100 μM in Chinese hamster ovary cells, to 230 μM in Novikoff hepatoma cells. In all cell lines investigated, the velocity of transport was sufficient to nearly completely equilibrate low concentrations of thymidine across the membrane within 15 s. Temperature dependence of transport velocity and substrate : carrier dissociation were continuous ($\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= 18.3}\text{kcal/mol}\text{, ΔH}{}^0\text{′ = 9.3}\text{kcal/mol}$, respectively), and showed no evidence of abrupt transitions. Several natural and artificial nucleosides and nucleic acid based inhibited influx of radiolabeled thymidine, apparently by competing with thymidine for the transport carrier.     

Participation of an active transport system in berberine-secreting cultured cells of Thalictrum minus

The release of the benzylisoquinoline alkaloid berberine from cultured cells of Thalictrum minus into the medium proved to be temperature-dependent and was suppressed by such inhibitors of the plasma membrane-bound ATPase as vanadate and diethylstilbestrol. These results indicate that berberine is secreted through an energy-requiring process located in the plasma membrane of berberine-producing T. minus cells. This is the first finding that a secondary metabolite of plant cell culture is secreted by an active transport system.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DES diethylstilbestrol - DMSO dimethyl sulfoxide - LS Linsmaier and Skoog (1965) - NAA 1-naphthaleneacetic acid     

Regulation of sugar transport in cultured diploid human skin fibroblasts

The regulation of hexose transport under glucose-starvation conditions was studied in cultured human skin fibroblasts. Glucose starvation enhanced the transport of 2-DG and 3-O-methyl-D-glucose (3-OMG) but not of L-glucose. Glucose-starvation enhanced transport was inhibited by cytochalasin B (10 μM). The starvation-induced change in 2-DG transport was due to an increase in the V_max of both the high and low affinity transport sites (2.8- and 2.4-fold, respectively) with no effect on their K_ms. The presence of 5.55 mM galactose, fructose, or L-glucose in the medium resulted in transport increases similar to those seen in glucose-starved cells, while the presence of 5.55 mM glucose, mannose, or 3-OMG repressed 2-DG transport. Glucose-starvation enhancement of 2-DG transport was blocked by cycloheximide (20 μg/ml) but not by actinomycin D (0.03 μg/ml) or α-amanitin (3.5 μM). Readdition of glucose (5.55 mM) for six hours to glucose-starved cells led to a rapid decrease in hexose transport that could be blocked by cycloheximide but not actinomycin D. Although readdition of 3-OMG to glucose-starved cells had little effect on reversing the transport increases, glucose plus 3-OMG were more effective than glucose alone. Serum containing cultures (10% v/v) of glucose-fed or glucose-starved cells exhibited rapid decreases in 2-DG transport when exposed to glucose-containing serum-free medium. These decreases were prevented by employing glucose-free, serum-free medium. The data indicate that hexose transport regulation in cultured human fibrob asts involves protein synthesis of hexose carriers balanced by interactions of glucose with a regulatory protein(s) and glucose metabolism as they affect the regulation and/or turnover of the carrier molecules.     

Hexose transport regulation in cultured hamster cells

Hamster (nil) cells maintained overnight in culture medium containing cycloheximide and either glucose or fructose exhibit strikingly different rates of hexose transport and metabolism (i.e., uptake). Pretreatment of cultures with sulfhydryl reagents makes it possible to determine initial transport rates for a physiological sugar such as galactose which is a catabolite in hamster cells. Using galactose transport as a model, hexose uptake enhancements can now be shown to be due almost entirely to increase in the rate of the transport step. The transport regulation can best be accounted for by a model comprised of 2 antagonizing mechanisms. This model involves turnover of transport carriers as well as inhibitory units (“regulators”). The experimental as well as the theoretical model may also apply to the well-known uptake enhancements observed in oncogenically transformed cells.     

Intracellular copper transport in cultured hepatoma cells

The distribution of copper in lysates prepared anaerobically from copper-resistant hepatoma cells radiolabeled with 67Cu was examined in pulse-chase experiments. Initially, the majority of the radioactivity (greater than 85%) coeluted with copper-metallothionein. As the chase time increased there was a gradual loss of 67Cu from metallothionein, with a concomitant increase in the level of 67Cu-labeled glutathione. There was also an increase in 67Cu incorporation into superoxide dismutase. These results suggest that the chelation of copper by metallothionein from a copper-glutathione complex (Freedman, J. H., Ciriolo, M. R., and Peisach, J. (1989) J. Biol. Chem. 264, 5598-5605) is a reversible process. Further, they demonstrate that the copper bound to metallothionein is not permanently sequestered, but can be incorporated into other copper proteins.     

Thymidine transport in cultured mammalian cells. Kinetic analysis, temperature dependence and specificity of the transport system.

The transport of thymidine has been characterized kinetically and thermodynamically in Novikoff rat hepatoma cells grown in culture and, less extensively, in mouse L cells, Chinese hamster ovary cells, P388 murine leukemia cells and HeLa cells. That the characterizations pertained to the transport system per se was ensured, (i) by employing recently developed methods for rapid sampling of cell/substrate mixtures in order to follow isotope movements within a few seconds after initial exposure of cells to substrate; (ii) by utilizing cells rendered, by genetic or chemical means, incapable of metabolizing thymidine; and (iii) by demonstrating conformity of the transport data to an integrated rate equation derived for a simple, carrier-mediated system. The results indicate that thymidine is transported into mammalian cells by a functionally symmetrical, non-concentrative system for which the carrier : substrate dissociation constant ranges from about 100 microM in Chinese hamster ovary cells, to 230 microM in Novikoff hepatoma cells. In all cell lines investigated, the velocity of transport was sufficient to nearly completely equilibrate low concentration of thymidine across the membrane membrane within 15 s. Temperature dependence of transport velocity and substrate : carrier dissociation were continuous (EA = 18.3 kcal/mol, delta H0' = 9.3 kcal/mol, respectively), and showed no evidence of abrupt transitions. Several natural and artificial nucleosides and nucleic acid bases inhibited influx of radiolabeled thymidine, apparently by competing with thymidine for the transport carrier.     

Regulation of plasmid-mediated iron transport and virulence inVibrio anguillarum

Summary Iron is essential for bacterial growth and metabolism. In vertebrates this metal is complexed by high-affinity iron-binding proteins, such as transferrin in serum. The fish pathogenVibrio anguillarum possesses a very efficient iron-uptake system which is encoded in the virulence plasmid pJMI. This allows the bacterium to utilize the otherwise unavailable iron in the fish host, resulting in the septicemic disease vibriosis. This system includes the siderophore anguibactin and transport components. We have cloned this iron-utpake system and have defined several genetic units by transposition mutagenesis. Nucleotide sequence analysis identified four open reading frames in the transport region, one of these corresponding to the gene for the outer membrane protein OM2 and another to a 40-kDa polypeptide. Complementation analysis indicated that products from all four reading frames are required for the transport of iron-anguibactin complexes. We have also identified positive and negative-acting regulatory elements that modulate in concert the expression of anguibactin biosynthetic genes and iron transport. The deletion or mutation of the positive-acting regulatory genes results in an iron-uptake-deficient phenotype and leads to an attenuation of virulence, underscoring the importance of this iron-uptake system as a virulence attribute ofV. anguillarum.     

Regulation of protein accumulation in cultured cells.

1. A technique is described whereby protein synthesis, protein breakdown and net protein accumulation are measured separately in monolayer cultures of mammalian cells. All rates are expressed as microgram of protein per 18 h incubation. 2. Under most incubation conditions with either L6 rat myoblasts or T47D human breast carcinoma cells the rates of protein accumulation, determined directly, agreed with the rates obtained by subtracting protein breakdown from protein synthesis. 3. Foetal calf serum, human and bovine colostrum, human milk and insulin increased protein accumulation in both cell lines, mainly as a consequence of effects on protein synthesis. 4. NH4Cl, in addition to inhibiting protein breakdown in both cell lines in the presence and in the absence of serum, stimulated protein synthesis in L6 myoblasts. 5. Leupeptin slightly inhibited protein breakdown without affecting protein-synthesis rates. 6. Cycloheximide almost completely inhibited protein synthesis, but restricted the net loss of cell proteins under most conditions because protein-breakdown rates were also decreased. 7. The assumptions, limitations and potential application of this technique for evaluating changes in protein turnover are described.     

Regulation of peptide amidation in cultured pituitary cells

The intermediate lobe of the pituitary contains the alpha-amidated peptide alpha-melanotropin and high levels of a copper and ascorbate-dependent peptidylglycine alpha-amidating monooxygenase (PAM) capable of converting peptides terminating in -X-Gly into amidated products (-X-NH2). As reported previously, the ability of cultured intermediate pituitary cells to produce alpha-amidated alpha-melanotropin declined rapidly. A decline in PAM activity assayed in vitro under optimized conditions failed to account quantitatively for the lack of production of alpha-amidated product, while a 100-fold decline in cellular levels of ascorbate could account for the lack of production of alpha-amidated product. Incubation of intermediate pituitary cultures with ascorbate partially restored the ability of the cells to produce alpha-amidated product without significantly increasing the level of PAM activity. In intermediate pituitary cultures made competent to produce alpha-melanotropin by addition of ascorbate, the actual extent of amidation occurring was modulated by the presence of specific secretagogues (bromocriptine or corticotropin-releasing factor). Cultured anterior pituitary cells showed a similar rapid 3-fold decline in PAM activity assayed in vitro under optimized conditions. Cellular levels of ascorbate also declined rapidly to levels 100-fold below those in the intact anterior pituitary. The addition of ascorbate to the anterior pituitary cultures rapidly restored the enzyme activity assayed in vitro to the levels in the initial cell suspension. Thus, production of amidated product peptide may be regulated by cellular levels of ascorbate, by cellular levels of PAM activity, and by the concentration of specific secretagogues to which the cells are exposed.