Rat liver adenyl cyclase activity in various thyroid states.

Thyroidectomized and euthyroid rats were injected with three doses of triiodothyronine (T(3)) or of the diluent over a 6 day period, and liver homogenates were assayed for basal, epinephrine-stimulated, and NaF-stimulated adenyl cyclase activity. Based on NaF-stimulated levels, total adenyl cyclase activity, expressed per milligram of liver protein, was increased after thyroidectomy. Administration of T(3) to either hypothyroid or euthyroid rats, however, had no effect on the NaF-stimulated levels. Basal and epinephrine-stimulated enzyme activities were the same in hypothyroid, euthyroid, and hyperthyroid (euthyroid + T(3)) liver homogenates. In contrast, injections of T(3) in hypothyroid rats increased the activities of basal and epinephrine-stimulated adenyl cyclase. In view of the findings in euthyroid and hyperthyroid liver, it is possible that this effect is transient. In general, no correlation was found between the effects of thyroid hormone on respiration and on adenyl cyclase activity of the rat liver. These results imply that the hepatic thermogenic response to thyroid hormone is not mediated by stimulation of adenyl cyclase activity with the possible exception of the early effects of T(3) in the athyroid rat.

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.

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.

Diabetes downregulates GLUT1 expression in the retina and its microvessels but not in the cerebral cortex or its microvessels.

Capillaries in the retina are more susceptible to develop microvascular lesions in diabetes than capillaries in the embryologically similar cerebral cortex. Because available evidence implicates hyperglycemia in the pathogenesis of diabetic retinopathy, differences in glucose transport into the retina and brain might contribute to this observed tissue difference in susceptibility to diabetes-induced microvascular disease. Thus, we compared levels of GLUT1 and GLUT3 expression in the retina, cerebrum, and their respective microvessels by Western blot analysis. In nondiabetic animals, the content of GLUT1 protein in retina and its microvessels was multifold greater than that of cerebral cortex gray matter and its microvessels. Streptozotocin-induced diabetes of a 2-week or 2-month duration reduced GLUT1 expression in the retina and its microvasculature by approximately 50%, but it resulted in no reduction in GLUT1 expression in cerebrum or its microvessels. The density of capillaries in retinas of diabetic animals did not change from normal, and so the observed decrease in GLUT1 expression in the retina and retinal capillaries of diabetic animals cannot be attributed to fewer vessels. Despite the diabetes-induced reduction of GLUT1 expression in retina, neural retina of diabetic rats still possessed more GLUT1 than the cerebrum. Retinal pigment epithelium (RPE) possessed more GLUT1 than neural retina or its microvessels, and expression of the transporter in the RPE was not affected by diabetes. GLUT3 levels were greater in cerebral gray matter than in retina, and they were unaffected by diabetes in either tissue. The effect of diabetes on GLUT1 expression differs between retina and cerebral cortex, suggesting that glucose transport is regulated differently in these embryologically similar tissues. Because diabetes results in downregulation of GLUT1 expression in retinal microvessels, but not in RPE, the fraction of the glucose entering the retina in diabetes is likely to be greater across the RPE than across the retinal vasculature.

The mechanism of the calorigenic action of thyroid hormone. Stimulation of Na plus + K plus-activated adenosinetriphosphatase activity.

In an earlier study, we proposed that thyroid hormone stimulation of energy utilization by the Na(+) pump mediates the calorigenic response. In this study, the effects of triiodothyronine (T(3)) on total oxygen consumption (Q(OO2)), the ouabain-sensitive oxygen consumption [Q(OO2)(t)], and NaK-ATPase in liver, kidney, and cerebrum were measured. In liver, approximately 90% of the increase in Q(OO2) produced by T(3) in either thyroidectomized or euthyroid rats was attributable to the increase in Q(OO2)(t). In kidney, the increase in Q(OO2)(t) accounted for 29% of the increase in Q(OO2) in thyroidectomized and 46% of the increase in Q(OO2) in euthyroid rats. There was no demonstrable effect of T(3) in euthyroid rats on Q(OO2) or Q(OO2)(t) of cerebral slices. The effects of T(3) on NaK-ATPase activity in homogenates were as follows: In liver +81% from euthyroid rats and +54% from hypothyroid rats. In kidney, +21% from euthyroid rats and +69% from hypothyroid rats. T(3) in euthyroid rats produced no significant changes in NaK-ATPase or Mg-ATPase activity of cerebral homogenates. Liver plasma membrane fractions showed a 69% increase in NaK-ATPase and no significant changes in either Mg-ATPase or 5'-nucleotidase activities after T(3) injection. These results indicate that thyroid hormones stimulate NaK-ATPase activity differentially. This effect may account, at least in part, for the calorigenic effects of these hormones.

Thyroid thermogenesis in adult rat hepatocytes in primary monolayer culture: direct action of thyroid hormone in vitro.

We have studied the effect of 3,5,3'-triiodothyronine (T3) on the respiration of adult rat hepatocytes in primary monolayer culture prepared from hypothyroid rat liver. After addition of T3 to the culture medium at a concentration of 2 x 10(-7) M, oxygen consumption of the cultured cells increased detectably at 24 h and was maximal at 72--96 h, relative to control cultures (38.0 +/- 1.8 vs. 25.0 +/- 1.5 microliter/h.mg protein). The thyroid-responsive enzymes, Na+ + K+-activated adenosine triphosphatase (NaK-ATPase) and alpha-glycerophosphate dehydrogenase (GPD), each exhibited increased activity in response to T3, in parallel with the change in oxygen consumption, whereas the activity of Mg-dependent ATPase was unaffected. These responses to T3 were dose dependent over similar concentration ranges, the half-maximal response for each occurring at ca 8 x 10(-10) M. In thyroid-treated cells, the observed increase in respiration was almost completely (90%) inhibited after addition of ouabain (10(-3) M) to the culture medium. It was found also that a 4-h exposure of the cultured hepatocytes to T3 was sufficient to elicit a significant thermogenic response, measured at a time (48 h later) when T3 was no longer present in the medium. The response to T3 occurred in fully defined culture medium and was independent of the presence or absence of hypothyroid rat serum, corticosterone, or insulin, and cellular ATP was unaffected by T3 in concentrations up to 2 x 10(-7) M. The findings document that adult rat hepatocytes in primary monolayer culture respond directly to thyroid hormone; the increases in respiration and NaK-ATPase activity elicited by T3 were cotemporal and apparently coordinate.

Stimulation of Na,K-activated adenosine triphosphatase and active transport by low external K+ in a rat liver cell line.

Exposure of ARL 15 cells, an established line from adult rat liver, to concentrations of external K+ below 1 mM caused a rapid fall in intracellular K+ and a corresponding rise in intracellular Na+ that became maximal within 12 h. Upon continued exposure to low external K+, these initial changes were followed by a striking recovery such that, by 24 h, intracellular Na+ and K+ concentrations approached their control values. Concomitant with this recovery, there was a substantial increase in Na,K-ATPase specific activity that was detectable at 12 h and maximal at 24 h. After restoration of the external K+ concentration, the elevated level of enzyme activity showed little change for at least 24 h. In contrast, restoration of external K+ resulted in a rapid rise in intracellular K+ and a fall in Na+ such that within 30 min the Na+/K+ ratio was lower than in control cells. This overshoot, together with a demonstrated increase in active 86Rb+ uptake under "Vmax" conditions, confirms that the enhancement in Na,K-ATPase specific activity in response to low external K+ represents an increase in functional Na,K pumping capacity.

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.

Effect of thyroid hormone on the abundance of Na,K-adenosine triphosphatase alpha-subunit messenger ribonucleic acid.

The effects of thyroid hormone on Na,K-ATPase alpha-subunit mRNA (mRNA alpha) content and Na,K-ATPase activity were measured in renal cortex, heart, and cerebrum of hypothyroid rats 24 and 72 h after injection of diluent or T3. Use of a cDNA probe complementary to rat brain mRNA alpha in Northern blot analysis revealed a single 26-27 S band in RNA isolated from these three tissues regardless of thyroid status. Tissue mRNA alpha content was estimated by dot blot analysis of whole cell extracts and isolated total RNA. Injection of T3 augmented mRNA alpha content by 2.1- to 2.5-fold in kidney cortex and myocardium at 24 h. After three daily injections of T3, the increases in mRNA alpha were evident despite a global increase in RNA content associated with hypertrophy of these target tissues. Furthermore, the increases in abundance of mRNA alpha after 72 h of T3 treatment correlated with enhancement of Na,K-ATPase activity. In contrast, both mRNA alpha and enzyme activity were invariant in the cerebrum. These data suggest that T3-induced augmentation of Na,K-ATPase activity is mediated, at least in part, by increased mRNA alpha content in target tissues.

Time course of Na,K transport and other metabolic responses to thyroid hormone in clone 9 cells.

To elucidate the relationship between the stimulation of Na+ and K+ fluxes by thyroid hormone and the induction of the Na,K-ATPase, we performed a detailed comparison of the time courses of these hormonal effects in a rat liver cell line, clone 9. Stimulations of passive K+ efflux, passive Na+ influx, and ouabain-inhibitable K+ uptake were all evident within 6-12 h of exposure of cells to T3 (10(-7) M). The time course of the induction of Na,K-ATPase activity closely paralleled that of the increase in the rate of Na+ and K+ fluxes. The maximal stimulatory effects of T3 on ouabain-inhibitable K+ uptake and Na,K-ATPase activity at 72 h were +49% and +36%, respectively. Intracellular Na+ and K+ contents were virtually unchanged during these increases in ion fluxes and Na,K-ATPase activity, suggesting an efficient homeostatic adaptation to the augmented passive "leak" of Na+ and K+ down their transmembrane concentration gradients. T3 treatment for 72 h was also shown to stimulate both lactate production (+62%) and [3H]2-deoxyglucose uptake (+82%) in these cells. The onset of these effects appeared to precede that of the stimulation of Na+ and K+ fluxes, being detectable at 4 h. Neither these latter effects of T3 nor the stimulation of ouabain-inhibitable K+ uptake could be demonstrated when RNA or protein synthesis was inhibited by actinomycin D or cycloheximide, respectively. It is concluded that in clone 9 cells thyroid hormone causes increases in passive Na+ influx, passive K+ efflux, active Na,K transport, and Na,K-ATPase activity whose time courses are closely parallel.

Stimulation of active Na+ and K+ transport by thyroid hormone in a rat liver cell line: role of enhanced Na+ entry.

A continuous cell line derived from rat liver (ARL 15) has been identified that responds to thyroid hormone with a stimulation of active Na,K transport. Stimulation of ouabain-inhibitable K+ uptake, which is half-maximal at a T3 concentration of 1.4 X 10(-10) M, is accompanied by corresponding increases in passive Na+ influx and in passive K+ efflux. The enhancement of Na+ and K+ fluxes by T3 is shown to be accompanied by smaller and equivalent increases both in enzymatically measured Na,K-ATPase activity and in maximal ouabain-sensitive Na,K transport in the presence of the Na+ ionophore monensin. The demonstration that both passive Na+ influx and passive fractional K+ efflux are simultaneously increased by T3 supports the earlier suggestion that the stimulation of active Na,K transport by thyroid hormone is attributable, at least in part, to an enhancement of rate-limiting passive Na+ and K+ fluxes by an increase in membrane permeability.

A variant of iodotyrosine-dehalogenase deficiency.

Three siblings (products of consanguineous marriage) affected with iodotyrosine-dehalogenase deficiency (presumed homozygotes) were found to have low thyroxine and large multinodular goiters, but none was mentally retarded. Iodide therapy corrected the serum T4 and thyroidal iodide uptake and discharge curve. The goiters shrank with iodide treatment. The subjects demonstrated significant ability to deiodinate intravenously injected L-mono-iodotyrosine (MIT) but not L-diiodotyrosine (DIT); 9.9% and 80.0% of an injected dose of 125I-MIT and 125I-DIT appeared unchanged in the urine 4 h, respectively. The data in presumed heterozygote subjects (both parents and two other siblings) were intermediate between controls and affected subjects. Thyroidal dehalogenase activity was measured in one of the affected subjects in vitro. The tissue showed greater ability to deiodinate MIT than DIT, but both activities were much lower than that of control tissue. The disease appears to be transmitted in an autosomal recessive fashion. The MIT-dehalogenase activity demonstrable in the affected individuals may explain the mild phenotype, in that MIT leaking from the goiter can be deiodinated to a significance degree and the liberated iodide reutilized.

Binding of zinc and iron to wheat bread, wheat bran, and their components.

Wholemeal wheat bread decreases the availability and intestinal absorption of divalent metals. To define this action further, binding of zinc in vitro to a wheat wholemeal bread (Tanok), dephytinized Tanok, and cellulose was determined at pH 5.0 to 7.5. Zinc binding by each was highly pH-dependent and reached a maximum at pH 6.5 to 7.5. Removal of phytate from Tanok did not reduce its binding capability. Wheat bran at pH 6.5 and 6.8 bound 72% of iron (0.5 microgram/ml of solution) and 82.5% of zinc (1.43 microgram/ml solution), respectively. Lignin and two of the hemicellulose fractions of wheat bran and high binding capabilities for zinc (85.6, 87.1, and 82.1%, respectively) whereas a third had a lower zinc-binding capability (38.7%). Binding of zinc to various celluloses and dextrans is also demonstrated. Formation of complexes of these metals with wheat fiber can explain, at least in part, the decreased availability of dietary iron and zinc in wholemeal wheat bread.

Folate content of Iranian breads and the effect of their fiber content on the intestinal absorption of folic acid.

Folate deficiency is a relatively uncommon disorder in central Iran. In order to explain this finding; the acid content of various Iranian breads was determined, since bread is the staple food in Iran. Tanok, the village wholemeal bread, has an average "free" folate content (without conjugase incubation) of 0.34 mug/g. Sangask and Bazari, leavened breads made from flours of high extraction rates and widely consumed in towns and cities, have aberage "free" folate contents of 0.38 and 0.71 mug/g, respectively. The folate content of these breads are significantly higher than that of white bread from refined flour (0.13 mug/g) or oatmeal bread (0.09 mug/g). Iranian breads also have a high content of indigestible fiber (1.6 to 4.2% of dry weight). Since substances within the bread, such as fiber, may interfere with folate absorption by the small intestine, sequential folate absorption tests (tritiated pterolymonoglutamic acid) were performed in four subjects with meals of increasing fiber content and fasting. No interference with folate absorption was found. Furthermore, in vitro studies did not demonstrate the formation of insoluble complexes between bread fiber and folic acid, which might indicate decreased availability.

Folate levels among various populations in central Iran.

Serum folic acid levels (Lactobacillus casei assay) were determined in 467 subjects representing various socioeconomic classes from rural and urban central Iran. Of the subjects 170 were pregnant females in different trimesters. The mean serum folate levels of the various groups studied ranged from 7.5 to 11.1 ng/ml and are within the normal reported range of 5.9 to 21.0 ng/ml. Only two subjects had serum folate levels below 3.0 to 5.9 ng/ml. In anemic subjects (hematocrit is less than 37%), red blood cell folate levels were also determined and were significantly higher than nonanemic controls (558 +/- 56 versus 346 +/- 35 ng/ml of packed red blood cells, P is less than 0.01). This difference may be related to underutilization of folate in patients with iron deficiency (a common metal deficiency in this area). A probable explanation for the well maintained serum folate levels in late pregnancy as well as in other populations studied in this report may be the high dietary intake of Iranian bread made from wheat flour of high extraction rate.

Evaluation of dynamic polar molecular surface area as predictor of drug absorption: comparison with other computational and experimental predictors.

The relationship between various molecular descriptors and transport of drugs across the intestinal epithelium was evaluated. The monolayer permeability (Pc) of human intestinal Caco-2 cells to a series of nine beta-receptor-blocking agents was investigated in vitro. The dynamic polar molecular surface area (PSAd) of the compounds was calculated from all low-energy conformations identified in molecular mechanics calculations in vacuum and in simulated chloroform and water environments. For most of the investigated drugs, the effects of the different environments on PSAd were small. The exception was H 216/44, which is a large flexible compound containing several functional groups capable of hydrogen bonding (PSAd,chloroform = 70.8 A2 and PSAd,water = 116.6 A2). The relationship between Pc and PSAd was stronger than those between Pc and the calculated octanol/water distribution coefficients (log Dcalc) or the experimentally determined immobilized liposome chromatography (ILC) retention. Pc values for two new practolol analogues and H 216/44 were predicted from the structure-permeability relationships of a subset of the nine compounds and compared with experimental values. The Pc values of the two practolol analogues were predicted well from both PSAd calculations and ILC retention studies. The Pc value of H 216/44 was reasonably well-predicted only from the PSAd of conformations preferred in vacuum and in water. The other descriptors overestimated the Pc of H 216/44 100-500-fold.

Regulation of glucose transport by hypoxia.

Transport of glucose into most mammalian cells and tissues is rate-controlling for its metabolism. Glucose transport is acutely stimulated by hypoxic conditions, and the response is mediated by enhanced function of the facilitative glucose transporters (Glut), Glut1, Glut3, and Glut4. The expression and activity of the Glut-mediated transport is coupled to the energetic status of the cell, such that the inhibition of oxidative phosphorylation resulting from exposure to hypoxia leads to a stimulation of glucose transport. The premise that the glucose transport response to hypoxia is secondary to inhibition of mitochondrial function is supported by the finding that exposure of a variety of cells and tissues to agents such as azide or cyanide, in the presence of oxygen, also leads to stimulation of glucose transport. The mechanisms underlying the acute stimulation of transport include translocation of Gluts to the plasma membrane (Glut1 and Glut4) and activation of transporters pre-exiting in the plasma membrane (Glut1). A more prolonged exposure to hypoxia results in enhanced transcription of the Glut1 glucose transporter gene, with little or no effect on transcription of other Glut genes. The transcriptional effect of hypoxia is mediated by dual mechanisms operating in parallel, namely, (1) enhancement of Glut1 gene transcription in response to a reduction in oxygen concentration per se, acting through the hypoxia-signaling pathway, and (2) stimulation of Glut1 transcription secondary to the associated inhibition of oxidative phosphorylation during hypoxia. Among the various hypoxia-responsive genes, Glut1 is the first gene whose rate of transcription has been shown to be dually regulated by hypoxia. In addition, inhibition of oxidative phosphorylation per se, and not the reduction in oxygen tension itself, results in a stabilization of Glut1 mRNA. The increase in cell Glut1 mRNA content, resulting from its enhanced transcription and decreased degradation, leads to increased cell and plasma membrane Glut1 content, which is manifested by a further stimulation of glucose transport during the adaptive response to prolonged exposure to hypoxia.

The effect of hypoxia on human trophoblast in culture: morphology, glucose transport and metabolism.

The response to hypoxia of trophoblast isolated from term placenta and maintained in culture was studied. Trophoblast exposed to normoxic (PO2 120-130 mmHg) or hypoxic (PO2 12-14 mmHg) conditions were examined by electron microscopy. After 48 h, the cytoplasm of the hypoxic cells was more electron-dense with increased numbers of mitochondria, lysosomes and vacuoles. Compared to normoxic cells, the surface microvilli of the hypoxic cells were sparse, short and unevenly distributed. [3H]thymidine incorporation by both hypoxic and normoxic trophoblast fell rapidly and equivalently after 2 days in culture. The percentage of cells with the proliferation-associated nuclear antigen, Ki 67, also decreased, but remained higher in hypoxic cells suggesting that hypoxia retarded completion of the cell cycle (normoxia, 10.80 +/- 2.51 s.e.; hypoxia, 19.87 +/- 2.73, P < 0.01). Glucose consumption was elevated in hypoxia (3.73 +/- 1.07 s.e. mumol/10(6) cells/24 h) as compared to normoxia (1.46 +/- 0.83, P = 0.01). Although lactate production was consistently higher in hypoxia, the difference was not statistically significant (hypoxia 5.38 +/- 1.54 mumol/10(6) cells/24 h versus normoxia, 1.52 +/- 0.29, P = 0.07). After 48 h, uptake of [3H]2-deoxglucose ([3H]2DG) by hypoxic cells was reduced to 12 per cent +/- 4.3 s.e. of that in normoxic cells; return to normoxia resulted in recovery within 10 min. Lineweaver-Burk plots of [3H]2DG uptake indicated high affinity (KM 2.2 +/- 0.4 x 10(-4) M) and low affinity transporters (KM 4.5 +/- 1.6 x 10(-3) M). Northern blot analysis identified mRNA for GLUT1 and GLUT3. In hypoxia, steady-state GLUT1 and GLUT3 mRNA were approximately three- and 10-fold higher than in normoxia respectively. Inhibitors of oxidative metabolism of glucose increased the uptake of [3H]2DG within 2 h, whereas hypoxia reduced uptake. Hence, trophoblast in culture survives in extreme hypoxia, but manifests striking changes in morphology and in glucose metabolism and transport. Completion of cell cycle appears to be retarded.

Stimulation of GLUT1 glucose transporter expression in response to inhibition of oxidative phosphorylation: role of reduced sulfhydryl groups.

Treatment of Clone 9 cells incubated in the absence of serum with 5 mM azide for 24 h results in an 8- and 3-fold induction in GLUT1 mRNA and GLUT1 protein, respectively. To explore the pathways mediating the induction of GLUT1 mRNA, we first examined whether inhibition of oxidative phosphorylation by other agents results to a similar response. Exposure of cells to 5 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP), 0.15 microM oligomycin B, or 5 mM azide resulted in near-equivalent increases in GLUT1 mRNA content. The inhibition of oxidative phosphorylation is associated with increased cell lactate content and in extracellular lactate to pyruvate ratio, reflecting a rise in cytosolic NADH/NAD+ ratio. We next tested the possibility that an increase in cell SH/SS ratio mediates the enhancement of GLUT1 mRNA in response to azide. We show that treatment of cells with 10 mM mercaptoethanol, an agent that increases cell SH/SS ratio, results in a approximately 6-fold increase in GLUT1 mRNA content. Moreover, incubation of cells in the presence of 0.3 mM diamide, a known intracellular sulfhydryl oxidizing agent, completely abolishes the induction of GLUT1 mRNA by azide. The results suggest that an increase in cell SH/SS ratio plays a critical role in the induction of GLUT1 mRNA in response to inhibition of oxidative phosphorylation.

Thyroid thermogenesis: minimal contribution of energy requirement for protein synthesis.

Cycloheximide (5.0 mug/ml) had no significant effect on respiration of liver slices prepared from euthyroid rats (i.e., QO2 of 15.5 +/- 0.8 vs. 14.9 +/- 0.8 mul/mg protein) despite an 83.8 +/- 2.1% inhibition of protein synthesis as judged by [3H]leucine incorporation into the trichloroacetic acid precipitable fraction. Injection of triiodothyronine increased the QO2 of rat liver slices to 22.6 +/- 1.1 mul/h/mg protein. The QO2 of hyperthyroid slices remained high in the presence of cycloheximide although protein synthesis was inhibited by 84.4 +/- 2.0%. These results imply that the energy requirement for protein synthesis contributes little to QO2 of euthyroid liver and does not account for a significant fraction of the increase in hepatic QO2 obtained in the transition from the euthyroid to the hyperthyroid state.