Faramarz Ismail-Beigi

Metabolic regulation of glucose transport

Facilitated transport of glucose across plasma membranes, a passive Na+-independent process present in all mammalian cells, is mediated by a family of homologous glycoprotein molecules that exhibit characteristic kinetic properties and are expressed in a tissue-specific manner. Because glucose is a universal energy-producing substrate, the regulation of its transport into cells is of fundamental importance in cellular homeostasis. This review is primarily focused on regulatory pathways that modulate the rate of glucose transport in response to alterations in cellular metabolism, with specific reference to conditions associated with increased demand for glucose utilization. Mechanisms mediating the regulation of glucose transport in response to a variety of other stimuli are also briefly considered.

Thyroidal enhancement of rat myocardial Na,K-ATPase: Preferential expression of α2 activity and mRNA abundance

SummaryIn hypothyroid rat myocardium, the low-ouabain-sensitivity Na,K-ATPase activity had aKi=10−4m and accounted for ∼95% of the enzyme activity, while the high-ouabain-sensitivity activity contributed ∼5% to the total activity, with aKi=3×107m. mRNAα1 was 7.2- and 5.5-fold more abundant than mRNAα2 and mRNAβ, respectively, in hypothyroid ventricles while mRNAα3 was undetectable. Administration of T3 increased total Na,K-ATPase activity 1.6-fold; the low-ouabain-sensitivity activity increased 1.5-fold while high-ouabain-sensitivity activity was stimulated 3.2-fold. T3 increased the number of high-affinity ouabain-binding sites 2.9-fold with no change inKd (∼2×10−7m). The abundances of mRNAα1, mRNAα2, and mRNAβ (per unit RNA) following T3 treatment increased 3.6-, 10.6-, and 12.7-fold, respectively. The larger increments in subunit mRNA abundances than in Na,K-ATPase activity suggests the involvement of translational and/or post-translational regulatory steps in Na,K-ATPase biogenesis in response to T3. It is concluded that T3 enhances myocardial Na,K-ATPase subunit mRNA abundances and Na,K-ATPase activity, and that the expression of the high- and low-ouabain-sensitivity activities are probably a reflection of the abundances of the α2 and α1 isoforms, respectively. The physiological role played by the β subunit remains uncertain.

Na,K-ATPase in several tissues of the rat: Tissue-specific expression of subunit mRNAs and enzyme activity

SummaryThe relative contents of Na,K-ATPase subunit mRNAs in rat renal cortex; ventricular myocardium, skeletal muscle (hind limb), liver and brain (cerebrum) were measured. Expressed per unit DNA, mRNAα1 content was ∼2-fold greater in the kidney and brain as compared to either heart, skeletal muscle or liver. The hierarchy of mRNAα2 expression was brain > skeletal muscle > heart, whereas mRNAα3 was restricted to brain. Betal subunit mRNA content in both kidney and brain exceeded the abundance of liver mRNAβ1 by ∼7-fold. In all tissues examined, the combined abundances of the alpha subunit mRNAs exceeded the content of mRNAβ1 The hierarchy of Na,K-ATPase activity expressed per unit. DNA was brain > kidney > skeletal muscle = heart > liver. The sum of mRNAα as well as mRNAβ1 content, expressed per g of tissue, was highest in brain and kidney. A statistically significant correlation between mRNAβ1 content and Na,K-ATPase activity was evident.

Thyroid hormone regulation of Na,K-ATPase expression

Active Na,K transport across plasma membranes (mediated by Na,K-ATPase) is stimulated by triiodothyronine (T(3)) in all mammalian tissues responsive to thyroid hormone, and this stimulation has been proposed to account for a substantial fraction of thyroid thermogenesis. The enhancement of Na,K-ATPase activity by T(3) results from increased biosynthesis of Na,K-ATPase subunits and is associated with increased abundance of their encoding mRNAs. In certain target tissues, T(3) preferentially augments the expression of the alpha2 isoform of the enzyme (characterized by its high sensitivity to inhibition by cardiac glycosides). The T(3)-induced increase in Na,K-ATPase subunit mRNA expression has been shown to be mediated by both transcriptional and post-transcriptional mechanisms.

Enhancement of Glucose transport in clone 9 cells by exposure to alkaline pH: Studies on potential mechanisms

SummaryIncubation of a nontransformed rat liver cell line. Clone 9, at pH 8.5 resulted in an ≈16-fold stimulation of cytochalasin B-inhibitable 3-O-methylglucose (3-OMG) transport, an effect that was independent of the presence of serum. Exposure to 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated 3-OMG uptake, and the enhancement was not additive to that produced by incubation at pH 8.5. In cells “depleted” of protein kinase C activity by a 20-hr exposure to TPA, however, the stimulation of 3-OMG transport in response to incubation at alkaline pH was still fully demonstrable. In control and alkaline pH-exposed cells, the inhibition of 3-OMG uptake by cytochalasin B was consistent with a single-site ligand binding model (K1≈10−7m). Northern blot analysis demonstrated the presence of only the human erythrocyte/rat brain/HepG2 cell glucose transporter-mRNA isoform (EGT), and the abundance of this mRNA was unchanged following exposure to alkaline pH. Immunoblot analysis, using polyclonal antibodies directed against the carboxy-terminal dodecapeptide of EGT, demonstrated and ≈2.0-fold increase in the abundance of transporters in partially purified plasma membrane fractions following incubation at pH 8.5, while EGT abundance was unchanged in whole-cell extracts. It is concluded that the stimulation of glucose transport in response to incubation of Clone 9 cells at alkaline pH does not require the presence of serum or activation of protein kinase C, and that the response is at least in part mediated by an increase in the number of glucose transporters in the plasma membrane.

OUABAIN-SENSITIVE NA+,K+-ATPASE ACTIVITY IN TOAD BRAIN

Toads of the genus Bufo are highly resistant to the toxic effects of digitalis glycosides, and the Na+,K(+)-ATPase of all toad tissues studied to date has been relatively insensitive to inhibition by digitalis and related compounds. In studies of brain microsomal preparations from two toad species, Bufo marinus and Bufo viridis, inhibition of ATPase activity and displacement of [3H]ouabain from Na+,K(+)-ATPase occurred over broad ranges of ouabain or bufalin concentrations, consistent with the possibility that more than one Na+,K(+)-ATPase isoform may be present in toad brain. The data could be fitted to one- or two-site models, both of which were consistent with the presence of Na+,K(+)-ATPase activity with high sensitivity to ouabain and bufalin. Ki (concentration capable of producing 50% inhibition of activity) values for ouabain in the one-site model were in the 0.2 to 3.7 microM range, whereas Ki1 values in the two-site model ranged from 0.085 to 0.85 microM, indicating that brain ATPase was at least three orders of magnitude more sensitive to ouabain than B. marinus bladder ATPase (Ki = 5940 microM). Ouabain was also an effective inhibitor of 86Rb+ uptake in B. marinus brain tissue slices (Ki = 3.1 microM in the one-site model; Ki1 = 0.03 microM in the two-site model). However, the relative contribution of the high ouabain-sensitivity site to the total activity was 17% in the transport assay as compared with 63% in the Na+,K(+)-ATPase enzymatic assay. We conclude that a highly ouabain-sensitive Na+,K(+)-ATPase activity is present and functional in toad brain but that its function may be partially inhibited in vivo.

Stimulation of glucose transport in Clone 9 cells by insulin and thyroid hormone: role of GLUT-1 activation

Thyroid hormone (T3) and insulin are both shown to stimulate glucose transport in Clone 9 cells, a rat liver cell line in which the utilization of glucose is limited by transport rate and in which only the GLUT-1 transporter isoform is expressed. Pre-treatment of these cells with T3 moreover substantially enhances the stimulatory effect of insulin such that at maximally effective hormone concentrations the effects of T3 and insulin on glucose transport are more than additive and indeed nearly multiplicative, suggesting that the mechanisms mediating the enhancement of glucose transport differ between the two hormones. Cell surface biotinylation followed by Western-blot analysis of plasma membrane fractions showed that the stimulatory effects of T3 and insulin on glucose transport, whether acting singly or in combination, exceed the attendant increases in the abundance of GLUT-1 in the plasma membrane. It is suggested that activation of GLUT-1 molecules pre-existing in the plasma membrane plays a major role in mediating the stimulatory effects of T3 and insulin on glucose transport in this cell line.

Role of enhanced Na+ entry in the control of Na,K-ATPase gene expression by serum

The role of enhanced Na+ entry in the induction of Na,K-ATPase subunit mRNAs by serum was investigated in a “nontransformed” rat liver cell line, Clone 9. Exposure of cells to 10% calf serum resulted in a 1.5-fold increase in the rate of Na+ entry associated with a transient rise in cell Na+ content (twofold at 15 min) and a sustained 1.15-fold rise in cell K+ content. After 6 hr of exposure to serum mRNAα1 and mRNAβ1 content increased by 1.8 and 2.6-fold, respectively. In nuclear run-on assays, serum stimulated the transcription of the α1 gene ∼ 1.9-fold while the transcription rate of the β1 gene remained unchanged. In cells incubated in Na+-free medium where NaCl was replaced by choline chloride, the induction of mRNAα1 by serum was fully preserved, whereas the increase in mRNAβ1 was prevented. An unexpected finding was that incubation of cells in Na+-free medium alone for 6 hr increased mRNAα1 but not mRNAβ1 content. These results indicate that Na,K-ATPase subunit mRNAs are differentially induced by serum, and that the induction of mRNAα1, in contrast to that of mRNAβ1, is transcriptionally mediated and does not require the presence of Na+ in the extracellular medium.

Thyroid hormone regulation of Na,K-ATPase subunit-mRNA expression in neonatal rat myocardium.

SummaryRegulation of Na,K-ATPase mRNAα isoform and mRNAβ expression by thyroid hormone (T3) in neonatal rat myocardium was examined. In euthyroid neonates between ages of 2 and 5 days, mRNAα1, mRNAα3, and mRNAβ1 abundances were nearly constant while mRNAα2 was undetectable. During the interval between postnatal days 5 and 15, mRNAα3 decreased to negligible levels and mRNAα2 became expressed and increased in abundance to account for ∼20% of the mRNAα pool by the 15th postnatal day. To examine the effect of T3 on this developmental program, neonates were injected with 75 μg T3/100 g body weight or diluent alone on the second and third postnatal days and myocardial Na,K-ATPase subunit-mRNA abundances were determined on the third and fourth postnatal days. Because T3 treatment increased the RNA/DNA ratios of myocardial tissue, the subunit-mRNA abundances were normalized per unit DNA. Following 24 and 48 hr of T3 treatment, the abundances of mRNAα1, mRNAα3, and mRNAβ1 increased, while mRNAα2 continued to remain undetectable during the 2-day interval between the second to fourth postnatal days. It is concluded that T3 augments the abundance of Na,K-ATPase subunit mRNAs that are already being expressed in the neonatal rat myocardium. The results further suggest that T3 does not act as a “molecular switch” in the developmental expression of the mRNAα isoforms in rat myocardium during the first four postnatal days.