Advances in our understanding of thyroid hormone action at the cellular level.
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Animals↗
Biomedical subjects
C N Mariash
Publications and source records attributed to C N Mariash.
Effect of thyroid hormones and high carbohydrate feeding on gene expression in rat epididymal adipose tissue.
We have determined the messenger RNA activity profiles of epididymal fat in euthyroid, hypothyroid, and hyperthyroid rats as well as in animals fed a high carbohydrate fat-free (lipogenic) diet. Radioautographs of two-dimensional gels of the in vitro translational products of RNA were quantitated by computer-assisted videodensitometry and analyzed by multivariate statistics. Of the 250 spots observed, each presumably representing the translational product of a separate messenger RNA, 21 were responsive to changes in hormonal state. Eighteen increased and two decreased at some stage in the transition from the hypothyroid to the hyperthyroid state. One spot changed in a biphasic manner. Eight products responded to the lipogenic diet, six increasing and two decreasing. Six of these eight spots responded in a similar fashion to thyroid hormone administration. As previously shown for liver, there appears to be a substantial overlap between the genomic response to carbohydrate and thyroid hormone administration. Whereas the carbohydrate-generated changes are largely included in the thyroid-hormone induced alterations, the majority of thyroid hormone responsive changes are not duplicated by the diet. Multivariate analysis showed a clear separation of each state from the others and revealed that it was necessary to monitor only nine spots to achieve an effective separation of the states analyzed.
Adipose Tissue↗
Revised nomenclature for tests of thyroid hormones and thyroid-related proteins in serum.
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Autoantibodies↗
Interaction of dietary carbohydrate and glucagon in regulation of rat hepatic messenger ribonucleic acid S14 expression: role of circadian factors and 3',5'-cyclic adenosine monophosphate.
The mRNA of the rat hepatic S14 gene accumulates rapidly after administration of T3 and carbohydrate, making it an excellent model for studies of the effects of dietary and hormonal stimuli at the hepatocellular level. We undertook studies to assess circadian changes in responsivity of this sequence to intragastric sucrose administration combined with insulin injection, and evaluated the capacity of glucagon to reverse these effects. As in the case of T3, the response of mRNA-S14 to carbohydrate in the morning was brisk whereas there was no significant increment when the stimulus was applied in the evening. In confirmation of previous studies, glucagon markedly lowered levels of mRNA-S14 in the evening but exerted no effect in the morning. These results support the concept that the rate of hepatic production of mRNA-S14 in unmanipulated rats is maximal in the evening, thus allowing no further induction by carbohydrate or T3 but permitting reduction by glucagon. Conversely, the rate of production is minimal in the morning, permitting induction by carbohydrate or T3 but allowing no further reduction by glucagon. A major difference between the effects of carbohydrate and those of T3 was the observed failure of carbohydrate to reverse the effect of glucagon in the evening. The effect of glucagon was stimulated by (Bu)2cAMP, and this was reversed by T3. However, T3 did not modify the glucagon-induced increase in hepatic cAMP levels. We therefore conclude that the capacity of T3 to abolish the glucagon effect is mediated at a step distal to the generation of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effects of insulin and glucose on cultured rat hepatocyte gene expression.
We previously used isolated adult rat hepatocyte cultures to study the ability of glucose to induce several hepatic mRNAs. However, we found that the optimal insulin concentration required to obtain the glucose effect was greater than 10,000 microU/ml. To test the hypothesis that the requirement for high concentrations of insulin in the culture was due to rapid loss of insulin in hepatocyte cultures, serial measurements of insulin were made at different media insulin concentrations (0-500,000 microU/ml) and glucose concentrations (5.5 and 2.75 mM). In addition, a dose-response relationship was established between media insulin concentrations and the pattern of mRNAs present in the hepatocytes determined by two-dimensional gel electrophoresis of in vitro translation products. We found that at low insulin concentrations (less than 1000 microU/ml), greater than 80% of the insulin was lost to the glassware, whereas at high initial insulin concentrations, approximately 23% of the insulin was lost to the glassware. Placement of media into the hepatocyte culture led to further insulin disappearance with a half-life for insulin of 41.5 h at 10,000 microU/ml and 13.8 h at 100 microU/ml. We found 16 mRNAs were altered by insulin at 5.5 mM glucose and 9 mRNAs were changed by insulin at 27.5 mM glucose. After taking into consideration the distributional and metabolic losses of insulin, all but one mRNA responded to insulin within the physiologic range of portal insulin (less than 1-94 microU/ml). Our data indicate that the hepatocyte culture is an excellent model to study the physiologic effects of insulin on hepatic gene expression.
Animals↗
Regulation of gene S-14 by triiodothyronine in liver.
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Animals↗
Revised nomenclature for tests of thyroid hormones and thyroid-related proteins in serum.
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Autoantibodies↗
Rapid synergistic interaction between thyroid hormone and carbohydrate on mRNAS14 induction.
Recent studies have shown that hepatic mRNAS14 responds rapidly to thyroid hormone administration. Moreover, this mRNA is known to increase in mass with the administration of a high carbohydrate fat-free diet. Therefore, it appears to share many of the same properties of the known hepatic lipogenic enzymes. Because the lipogenic enzymes display a synergistic interaction between thyroid hormones and carbohydrates, we investigated the kinetics of response of mRNAS14 to carbohydrate feeding, as well as its interaction with triiodothyronine (T3). We found that mRNAS14 responds rapidly to the dietary administration of sucrose in euthyroid rats, with a 2-fold increase within 30 min, and a 25-fold increase by 4 h. On the other hand, when given to hypothyroid rats, sucrose ultimately lead to only a 2-3-fold increase in the level of mRNAS14, attaining a level less than that found in starved euthyroid rats. The diminished response of mRNAS14 to sucrose in hypothyroidism could not be enhanced by insulin administration. However, administration of replacement doses of T3 (400 ng/100 g of body weight) immediately restored the rapid response to sucrose feeding. The response of sucrose and T3 was synergistic. Dose-response studies with T3 indicated that the rapid interaction between T3 and sucrose was limited by the occupancy of the T3 nuclear receptor. A similar synergistic response to T3 and glucose was noted in primary hepatocyte cultures, thus indicating that the synergism between these two stimuli is not due to changes in extrahepatic hormones or metabolites. Our data are most consistent with the hypothesis that the T3-nuclear receptor complex multiplies a signal generated by carbohydrate metabolism to induce hepatic mRNAS14. The interaction does not appear to require the preliminary induction of carbohydrate-metabolizing enzymes and their mRNAs.
Animals↗
Effect of dichloroacetic acid on rat hepatic messenger RNA activity profiles.
We have previously suggested that the ability of glucose to induce rat hepatic lipogenic enzymes is mediated by the mitochondrial oxidation of pyruvate. In part, this hypothesis is supported by the finding that an activator of pyruvate dehydrogenase, dichloroacetic acid (DCA), is capable of inducing malic enzyme in hepatocyte cultures. In order to further test this hypothesis, we compared the mRNA responses induced by carbohydrate feeding in vivo and by glucose administration to hepatocytes in culture with those mRNA responses induced in DCA both in vivo and in culture. DCA administration to rats resulted in a significant increase in liver:body weight ratio. It was, in addition, a potent inducer of malic enzyme. Hepatic mRNA activity profiles were examined by two-dimensional gel electrophoretic analysis of in vitro translation products. Six of the seven mRNAs altered by carbohydrate feeding were similarly altered by DCA feeding in vivo. In cultured hepatocytes 10 mmol/L DCA significantly increased four of six glucose-induced mRNAs. The mRNA for malic enzyme was among those mRNA sequences induced both in vivo and in culture. Increasing glucose concentrations in the culture medium resulted in an expected rise in pyruvate levels, whereas DCA caused a significant decrease in the concentration of this intermediate. It is likely, therefore, that augmentation of the flux of pyruvate through pyruvate dehydrogenase rather than alterations in pyruvate levels per se, is a proximal event leading to the induction of multiple mRNAs. The marked overlap in mRNA response to both carbohydrate and DCA indicates that the signal regulating the content of the carbohydrate responsive mRNAs is derived from mitochondrial pyruvate oxidation.
Acetates↗
Regulation of hepatic gene expression by lipogenic diet and thyroid hormone.
Adaptive hyperlipogenesis in the liver occurs after feeding rats a high-carbohydrate, fat-free diet or after treatment with thyroid hormone. This phenomenon is accompanied by the induction of a family of enzymes and proteins involved in various aspects of lipogenesis, resulting largely from alterations in the rates of protein synthesis. The changes in protein production, in turn, are the result of increased or decreased cellular concentrations of specific mRNAs in response to carbohydrate feeding or hyperthyroidism. There is a large degree of overlap between the mRNA species induced by thyroid hormone and carbohydrate feeding in rat liver. One such mRNA species, spot 14, has several attractive features as a potential model for exploring the molecular mechanisms involved in the regulation of gene expression. Most importantly, the mRNA for spot 14 increases with a lag time of less than 20 min after treatment with thyroid hormone or sucrose gavage and thus may represent a primary response to the hormonal and dietary stimuli. Initial studies to elucidate the cellular site of action of thyroid hormone and dietary carbohydrate indicate that changes in both the rate of gene transcription and the stability of the nuclear RNA precursor are involved in spot 14 mRNA induction.
Animals↗
Interaction of thyroid hormone and nutritional signals on thyroid hormone action.
The interaction between thyroid hormone (T3) and nutritional signals has been of interest for nearly a century. Thus, enhanced glucose production, absorption and utilization are associated with hyperthyroidism, whereas diminished glucose utilization and lipogenesis characterize hypothyroidism. Recent studies have uncovered what appears to be yet another area of interaction at the molecular level. On the one hand, a marked overlap exists between the changes in rat hepatic mRNA activity profile induced by hyperthyroidism and high carbohydrate administration. On the other hand, the patterns produced by hypothyroidism, starvation and diabetes are characterized by oppositely directed shifts. These findings may be due, in part, to a synergistic relationship between carbohydrate feeding and T3 administration in the induction of many hepatic lipogenic enzymes and their respective mRNAs. Studies both in the intact rat as well as in isolated hepatocyte cultures indicate that this synergism arises from the ability of T3 to multiply an intracellular signal derived from the metabolism of glucose. The development of recombinant DNA techniques can now be applied to the study of the interaction of T3 with nutritional signals. Initial efforts have demonstrated a hepatic mRNA (mRNAS14) rapidly responsive to both T3 and carbohydrates. With this probe, studies are under way to define the precise molecular mechanisms by which T3 and carbohydrates interact to influence gene expression.
Animals↗
Hepatic messenger ribonucleic acid activity profile of rats subjected to alterations in thyroidal and adrenocortical states: evidence for significant interaction.
We have previously established the value of 2-dimensional electrophoretic mRNA activity profiles for investigating the hepatic genomic response to several metabolic perturbations, such as thyroid hormone or GH treatment, diabetes, high carbohydrate diet, starvation, and uremia. We now report the effects of adrenalectomy and dexamethasone treatment, and compare these with alterations due to thyroidectomy and T3 treatment. Total rat hepatic RNA was isolated and translated in a reticulocyte lysate system. The [35S]methionine-labeled translated products were separated by 2-dimensional gel electrophoresis and quantified with computerized videodensitometry. Of 200 consistently quantifiable products, 14 (7%) were altered by adrenalectomy and dexamethasone, including 4 products (46, 47, 56, and 57) which have not been observed to change in previous studies from this laboratory. Adrenalectomy increased 5 and decreased 2 products, whereas dexamethasone increased 1 and decreased 8 products. Two products maintained the same directional shift in the transitions form adrenalectomy to control and from control to the dexamethasone-treated state. Thyroidectomy and T3 altered 13 products. Thyroidectomy increased 2 and decreased 7 products, whereas T3 treatment increased 6 and decreased 3 products. Four products maintained the same directional shift in the transitions from thyroidectomy to control and from control to the T3-treated state. In all of the manipulations performed (adrenalectomy, thyroidectomy, dexamethasone treatment, and T3 treatment), a total of 20 separate products changed. One third were affected by alterations of both the steroidal and thyroidal states. However, when adrenalectomy and thyroidectomy were compared, only 7% of the shifts were concordant, whereas 30% of the shifts were concordant when treatment with dexamethasone and T3 were compared. These results demonstrate that the mRNA activity response is highly specific for each hormonal manipulation. In addition, unanticipated interrelationships between steroidal and thyroidal states were observed. In some, the presence of T3 appears necessary for the suppressive effect of dexamethasone. Others show that T3 appears to inhibit a stimulatory effect of dexamethasone. Specificity of response to dexamethasone is emphasized by the lack of response to vitamin D, deoxycorticosterone, and dihydrotestosterone and by a different response to estradiol from dexamethasone.
Adrenal Cortex↗
Transcellular and transnuclear transport of 3,5,3'-triiodothyronine in isolated hepatocytes.
We have recently reported evidence for the presence of stereospecific energy-dependent transport processes for T3 in rat tissues. These processes were responsible for maintenance of concentration differences of free L- and D-T3 across the cellular plasma and nuclear membranes. In rat liver, the free L-T3 concentration in cytosol was almost 3 times higher than that in plasma, and nuclear free L-T3 was 58-fold that in cytosol. In the present studies, freshly isolated hepatocytes were used to study these processes in vitro. Kinetic experiments demonstrated that equilibrium of [125I]T3 between cells and medium was rapid and complete within 5 min. Neither the rate of cellular accumulation nor the equilibrium distribution of T3 between cells and medium was influenced by the addition of up to 2 X 10(-7) M T3. Equilibrium of T3 between the nuclear and extranuclear fractions of the hepatocytes was reached more slowly, only after 45-60 min of incubation. The nuclear free T3 concentration was calculated from mass action principles with knowledge of the association constant (Ka) of the nuclear T3-binding sites under in vitro conditions and the fractional occupancy of the sites. Cytosolic free T3 was determined from measurements of the fraction of cellular [125I]T3 associated with cytosol (pc), and the binding power of cytosol was determined by equilibrium dialysis (bc). The cytosol to plasma free T3 ratio in these cells was near unity, suggesting an absence of the concentration difference previously observed in liver in situ. The nuclear to cytosol free T3 ratio was 7.9, approximately 7 times less than that in vivo. The addition of 2 mM KCN caused a further 23% reduction in the nuclear to cytosol ratio. As previously reported for liver in situ, uptake of T3 by hepatocytes is stereospecific. Cellular uptake of D-T3 was greater than that for L-T3. However, nuclear transport favored L-T3. The nuclear to cell ratio for L-T3 was almost 4 times greater than that for D-T3 (mean +/- SEM, 0.020 +/- 0.0005 vs. 0.0085 +/- 0.0005; P less than 0.001). Our studies indicate the presence in the isolated hepatocyte of a nuclear transport process for T3 similar to that observed in vivo, but operating with a markedly reduced efficiency.
Animals↗
Use of the dexamethasone-adrenocorticotropin test to assess the requirement for continued glucocorticoid replacement therapy after pituitary surgery.
We assessed the need for continued glucocorticoid replacement therapy in postsurgical pituitary tumor patients using a dexamethasone-ACTH test. The patients received 1 mg dexamethasone, orally, at 2300 h and 250 micrograms synthetic ACTH (Cosyntropin), iv, at 0800 h the next morning. The mean +/- SD integrated cortisol response for a 2-h period of the 31 pituitary tumor patients [1264 +/- 924 micrograms X min/dl (34.87 +/- 25.49 mumol X min/liter)] was significantly less (P less than 0.005) than that of 25 normal subjects [3331 +/- 544 micrograms X min/dl (91.90 +/- 17.04 mumol X min/liter)]. Replacement glucocorticoids were abruptly discontinued in 11 patients with responses above 1450 micrograms X min/dl (40.01 mumol X min/liter). No clinical or laboratory evidence of adrenal insufficiency occurred as long as 15 months after discontinuation. Metyrapone tests, however, in the 11 glucocorticoid-withdrawn patients revealed a reduced mean +/- SD serum 11-deoxycortisol level compared with that of 10 normal subjects [8.9 +/- 4.7 vs. 15.6 +/- 5.0 micrograms/dl (0.26 +/- 0.13 vs. 0.45 +/- 0.16 mumol/liter); P less than 0.005]. Our results indicate that the dexamethasone-ACTH test is useful in identifying patients in whom replacement glucocorticoid therapy can be safely withdrawn under nonstressed conditions. The test can be simplified to one plasma cortisol level determined 30 min after ACTH administration.
Adrenocorticotropic Hormone↗
T3 stimulates the synthesis of a specific mRNA in primary hepatocyte culture.
The mechanism of triiodothyronine (T3) induction of a thyroid hormone responsive mRNA (mRNAS14) was studied in primary cultures of adult rat hepatocytes. T3 induced mRNAS14 in less than one hour after addition to the cultures. After 24 hours exposure to T3, the level of mRNAS14 was 2.5 to 6 times above the untreated controls. Addition of Actinomycin-D to both induced and control cultures led to similar mRNAS14 disappearance curves, implying that T3 augments the synthesis of mRNAS14 rather than stabilizing pre-formed mature mRNA. Glucagon inhibits the T3 induction of mRNAS14. When added to both induced and control cultures, glucagon leads to similar fractional decay curves for mRNAS14, confirming the Actinomycin-D studies. These findings demonstrate T3 induces mRNAS14 directly in the hepatocyte by increasing the synthesis of the mature mRNA.
Animals↗
Comparison of effectiveness of thyrotropin-suppressive doses of D- and L-thyroxine in treatment of hypercholesterolemia.
In an attempt to compare the cholesterol-lowering effects of equivalent doses of D- and L-thyroxine, 10 euthyroid, hypercholesterolemic subjects were treated with graded doses of each medication in a cross-over design using thyrotropin suppression following thyrotropin-releasing hormone administration as the end-point. The mean thyrotropin-suppressive dose of D-thyroxine was 2.4 +/- 0.66 mg per day, which resulted in mean reductions of 10 percent in total plasma cholesterol, 10 percent in plasma low-density lipoprotein cholesterol, and 11 percent in plasma high-density lipoprotein cholesterol. The mean thyrotropin-suppressive dose of L-thyroxine was 135 +/- 46 micrograms per day, which resulted in mean reductions of 7 percent in total plasma cholesterol, 6 percent in plasma low-density lipoprotein cholesterol, and 14 percent in plasma high-density lipoprotein cholesterol. The reductions in total, low-density, and high-density cholesterol achieved with D-thyroxine were not significantly different from those achieved with L-thyroxine. Neither medication produced a significant increase in heart rate or ventricular ectopy as determined by Holter monitoring. These data do not support the belief that D-thyroxine has a preferential cholesterol-lowering effect in humans when compared with equivalent doses of L-thyroxine. In addition, both D- and L-thyroxine reduced plasma high-density lipoprotein cholesterol.
Adult↗
Stimulation of malic enzyme formation in hepatocyte culture by metabolites: evidence favoring a nonglycolytic metabolite as the proximate induction signal.
Recent studies have shown that the addition of increasing concentrations of glucose to the medium of primary adult rat hepatocyte cultures results in the progressive induction of malic enzyme. We have undertaken experiments to determine (1) whether metabolism of glucose was an essential prerequisite for such induction, and (2) whether a specific glycolytic intermediate could be shown to constitute the proximate carbohydrate signal triggering such induction. In line with these objectives we investigated the ability of various sugars and glycolytic metabolites to induce malic enzyme in this system and assessed the influence of insulin, glucagon, and thyroid hormone (triiodothyronine, T3) on this process. Our results show that only those sugars capable of entering the cell and being metabolized induce malic enzyme (glucose, fructose, and galactose). The nonmetabolizable sugars 3-O-methylglucose and 2-deoxyglucose are ineffective. Incubation with 20 mmol/L lactate, pyruvate, dihydroxyacetone, or glycerol resulted in malic enzyme induction, whereas incubation with acetate, citrate, and alpha-ketoisocaproate was without effect. The induction by all sugars and metabolites required presence of insulin. As previously reported for glucose, addition of T3, under all metabolic conditions, resulted in a constant 3.6-fold increase in the rate of malic enzyme induction and further supports the proposal T3 acts to multiply the effect of a common carbohydrate-generated signal. Glucagon administration led to a dose-dependent inhibition of the carbohydrate effect with a half-maximal effect and maximal effect at 2 and 100 nmol/L, respectively. None of the glycolytic metabolites tested could reverse the glucagon inhibition completely.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Quantitative investigation of hepatic genomic response to hormonal and pathophysiological stimuli by multivariate analysis of two-dimensional mRNA activity profiles.
We have applied techniques of multivariate analysis to the characterization and comparison of the effects of various pathophysiological and hormonal stimuli on the expression of the rat hepatic genome at a pretranslational level. In vitro translated products were resolved by two-dimensional gel electrophoresis. We analyzed 10 pathophysiological states brought about by variation in thyroidal status, starvation, administration of high carbohydrate diet, and the production of experimentally induced diabetes mellitus. Each state differed significantly from every other state in the two-dimensional electrophoretic profiles. The set consisting of the minimal number of products necessary for maintaining the distinctive patterns was identified. The analysis also defined those clusters of products that behaved in a coordinate fashion in response to the various stimuli. Lastly, the similarity and dissimilarity of hepatic mRNA activity profiles to each other could be geometrically represented in three-dimensional space. Our finding that the hepatic mRNA activity profile could distinguish reliably between closely related hormonal and pathophysiological stimuli indicates the specificity of hepatic genomic expression. A systematic analysis of such profiles may be useful as an overall index of the biologic response at the hepatocellular level.
Animals↗