Insulin-mediated inhibition of the induction of tyrosine aminotransferase by dexamethasone.

Insulin-mediated regulation of glucocorticoid-induced expression of the liver-specific gene tyrosine aminotransferase was studied in a clone of the Reuber rat hepatoma cells. Insulin inhibited dexamethasone-induced chloramphenicol acetyltransferase expression from approximately 4 kb of TAT 5' flanking sequence. The degree of this inhibition was comparable to the response of the endogenous gene. A construct of approximately 3 kbp of 5' flanking sequence exhibited no significant basal expression but retained sensitivity to glucocorticoids and to insulin inhibition of the glucocorticoid response. Results of further analysis of the insulin response in deletion constructs and constructs containing glucocorticoid responsive elements ligated to a heterologous promoter suggest that in addition to the glucocorticoid response elements a region close to the start site in the TAT promoter is necessary for insulin to inhibit glucocorticoid-mediated induction of expression.

Nanomolar cyclic adenosine and guanosine monophosphates stimulate macrophage colony-stimulating factor responsiveness by murine transitional progenitors.

Both 3':5' cyclic adenosine monophosphate (cAMP) and 3':5' cyclic guanosine monophosphate (cGMP) stimulated colony-stimulating factor 1 (CSF-1)-dependent colony formation by murine two-signal-dependent progenitors without influencing colony formation by committed CSF-1-responsive progenitors. The stimulatory effect was optimal at 10(-9) M and did not diminish with increasing concentrations of the cyclic nucleotides. The membrane-permeating analogs dibutyryl cAMP and 8-Br-cGMP similarly augmented colony formation by the transitional progenitors at 10(-9) M; however, with increasing concentration, enhancement diminished with eventual inhibition of total colony formation at micromolar concentrations. Stimulation by the two cyclic nucleotides was mutually incompatible. The results indicate that physiological levels of extracellular cyclic nucleotides may significantly influence myelopoiesis. Furthermore, the results introduce the interesting possibility that stimulation, unlike inhibition, may be initiated through an extracytoplasmic mechanism that does not require direct activation of cytoplasmic cyclic nucleotide-dependent protein kinases.

An adaptive aortic pressure observer for the Penn State Electric Ventricular Assist Device.

This paper addresses the development of an aortic pressure observer for the Penn State Electric Ventricular Assist Device (EVAD). The observer estimates the aortic blood pressure by measuring the voltage of the electric motor and the pusher plate position. The estimated pressure is fedback to the EVAD's blood flow controller, which adjusts the beat rate of the device to accommodate the varying demand of cardiac output. The gains of the observer are deterministically optimized such that the optimal values are independent of the (often unknown) system state initial conditions. To improve the performance of the pressure observer an adaptation scheme is developed. In this scheme the initial pressure estimate of the succeeding systolic cycle is adjusted when the pressure does not match its corresponding second estimated value. In vitro test runs of the developed observer show that is is robust to parameter variations, and the error of the resultant estimated pressure is less than 5%.

Insulin-mediated regulation of tyrosine aminotransferase in rat hepatoma cells: inhibition of transcription and inhibition of enzyme degradation.

Insulin induces the enzyme tyrosine aminotransferase (TAT) in Reuber H-35 rat hepatoma cells. A clone of these cells (KRC-7) was used to study the relationship between changes in enzyme activity and hybridizable mRNA, and rates of transcription for TAT in response to insulin. Our results indicate that enzyme activity is inducible by insulin in the presence of an inhibitor of RNA synthesis, suggesting that insulin functions post-transcriptionally to increase enzyme activity. Unexpectedly, insulin causes a decrease in the level of hybridizable TAT mRNA. Glucocorticoids cause an increase in TAT mRNA and insulin inhibits this increase when added either subsequent to or simultaneous with the addition of this agonist. Transcriptional runoffs demonstrate that insulin inhibits transcription of TAT to account for the aforementioned decrease in hybridizable mRNA. To examine the possibility that a post-translational mechanism is responsible for the increase in TAT activity caused by insulin, the rate of degradation of TAT protein was measured using polyclonal antibody. These experiments indicate that the rate of degradation of TAT is decreased about twofold in the presence of insulin, which suggests that part of the observed increase in TAT activity is due to selective post-translational stabilization of TAT. Therefore, insulin regulates TAT in KRC-7 cells by both transcriptional and post-translational mechanisms, the latter being responsible for the increase in activity.

Regulation of tyrosine aminotransferase by insulin and cyclic AMP: similar effects on activity but opposite effects on transcription.

Tyrosine aminotransferase (TAT) is a liver-specific enzyme whose activity is subject to positive regulation by several agents including insulin and agonists that increase the intracellular concentration of cAMP. To further characterize the mechanism of insulin action and the interaction between cAMP and insulin several types of experiments were performed in a rat hepatoma cell-line. In the presence of the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranoyslbenzimidazole, TAT enzyme activity remains inducible by insulin and to a lesser extent by the cAMP analog (Bu)2cAMP. This suggests that transcriptional events are not necessary for the insulin-mediated increase in TAT activity, and also suggests a dualistic mechanism for the cAMP-induced increase in TAT activity. Surprisingly, using a cDNA probe for mRNATAT, it was found that insulin causes a decrease in hybridizable mRNATAT, in addition to causing a partial inhibition of the increase of hybridizable TAT transcript caused by (Bu)2cAMP. Examination of the rate of transcription of the TAT gene by a nuclear run-off assay shows that insulin causes a decrease in the transcription of the TAT gene by greater than 50%, which is sufficient to account for the decrease in hybridizable mRNATAT. As expected (Bu)2cAMP increases the transcription of TAT, but combined with insulin a complete inhibition of the increase in TAT transcription caused by (Bu)2cAMP is observed. To address the possibility that insulin acts posttranslationally to increase TAT activity, the t1/2 of TAT protein was measured in the presence and absence of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotensin regulation of macrophage colony-stimulating factor-stimulated in vitro myelopoiesis.

Neurotensin, at less than or equal to 10(-9) M, in the presence of an optimal concentration of macrophage CSF (CSF-1), stimulated a dose-dependent enhancement of colony formation by murine marrow-derived mononuclear phagocyte progenitor cells. The additional colonies arose from the cell cycle and Ia Ag-positive subpopulation previously identified as two-signal-dependent progenitors. Two-signal colony formation diminished when the peptide was added at concentrations greater than 10(-9) M. Neurotensin binds specifically to two distinct receptors, a high affinity receptor (KD approximately 10(-9) M) and a lower affinity (KD approximately 10(-7) M) receptor identified as the tuftsin receptor. Rat liver ferritin and an inhibitory tuftsin analog. (ALA1)-tuftsin, which inhibit two-signal colony formation stimulated by tuftsin and tuftsin-like peptides in combination with CSF-1, did not inhibit colony formation stimulated by CSF-1 and 10(-9) M neurotensin. Both inhibitors, however, reversed the loss of two-signal colony growth in the presence of higher neurotensin concentrations. Neurotensin fragment 1-6, unlike ferritin and (ALA1)-tuftsin, inhibited two-signal colony formation stimulated by 10(-9) M neurotensin. However, like ferritin and (ALA1)-tuftsin, fragment 1-6 permitted full expression of two-signal colony formation in the presence of CSF-1 and 10(-7) M neurotensin. The data indicated that occupancy of both receptors at neurotensin concentrations greater than 10(-9) M might be responsible for the diminished progenitor response. The data further support a potential role for neurotensin as an inflammatory mediator. In addition to direct effects on mature phagocytic leukocytes, neurotensin, at least in vitro can influence the production of new mononuclear phagocytes.

Insulin and phorbol myristic acetate induce ornithine decarboxylase in Reuber H35 rat hepatoma cells by different mechanisms.

Reuber H35 rat hepatoma cells respond to insulin or to tumor promoting phorbol esters with an increase in ornithine decarboxylase enzyme activity. This occurs in a time- and dose-dependent manner with both types of agonist. We report here that the increase in ornithine decarboxylase activity with optimal concentrations of both agonists is additive. Furthermore, the initial increase is dependent on continued RNA and protein synthesis. We also find that both of these agonists cause an increase in mRNA coding for ornithine decarboxylase in a time- and dose-dependent manner which suggests that the increase in enzyme activity can be accounted for by the increase in transcript levels. The difference in the time course of induction by the agonists, the additivity of induction by the two agonists, the differential sensitivity of induction to cycloheximide and RNA synthesis inhibitors, and the observation that phorbol myristic acetate causes a further increase in ornithine decarboxylase activity and transcript levels in cells already maximally induced by insulin suggest that these two agonists act through separate mechanisms.

Phorbol ester inhibition of hormonal induction of tyrosine aminotransferase.

The liver specific enzyme, tyrosine aminotransferase, can be induced by glucocorticoids, cAMP analogs, or insulin. Each of these different inducing agents is believed to act through a separate pathway. The tumor promoting phorbol esters have been reported to stimulate phosphorylation of the insulin receptor and thereby decrease the ability of insulin to induce tyrosine aminotransferase. Our results demonstrate that TPA will not only inhibit the insulin stimulated increase in tyrosine aminotransferase, but will also inhibit induction of the enzyme by glucocorticoids or by cAMP.

Dexamethasone permits the release of an inhibitor of pyruvate dehydrogenase from Reuber H-35 hepatoma cells in response to insulin.

Reuber H-35 rat hepatoma cells respond to physiological levels of insulin as a growth factor. Glucocorticoids antagonize this response. A chemical mediator of insulin action which activates mitochondrial pyruvate dehydrogenase has also been isolated from these cells. The present report demonstrates that if the H35 cells are incubated with glucocorticoid before treatment with insulin, they produce not only the stimulator, but also inhibitory mediator. Cells exposed to the glucocorticoid but not to insulin do not produce the inhibitory mediator. Therefore, insulin interaction with the cell is necessary to elicit this negative modifier of pyruvate dehydrogenase. A time course of the response suggests that the effect of the glucocorticoid is time dependent. The inhibitory mediator can be separated from the stimulatory mediator by molecular sieve chromatography. These results suggest a biochemical basis for glucocorticoid-mediated insulin resistance.

The role of the insulin receptor in mediating the insulin-stimulated growth response in Reuber H-35 cells.

Insulin is able to stimulate a growth response in a variety of different cell types. However, the role of the insulin receptor in mediating this response is not clear. Indeed, it has been reported that the ability of insulin to stimulate a growth response is a result of its interaction with other growth factor receptors rather than the insulin receptor. We have previously reported that the H-35 hepatoma cell line responded to physiological concentrations of insulin as a growth factor and that the relative potency of proinsulin suggested that this response was mediated by the insulin receptor. In this report, two experimental approaches are used to demonstrate the involvement of the insulin receptor in mediating the growth response. Two different preparations of antibody to the insulin receptor are found to be capable of stimulating this response. In addition, the human insulin-like growth factors (IGF-I and II) show very low cross-reactivity with the insulin receptor and are significantly less potent than insulin in stimulating the growth response.

Stimulation of tyrosine aminotransferase degradation by methylthioinosine.

Methylthioinosine (MeSno) is a purine nucleoside analog which is cytotoxic to a number of cultured cell lines including the Reuber H35 hepatoma cells used in the present studies. It has also been observed to cause a rapid profound loss of tyrosine aminotransferase activity in H35 cells well before the onset of any measurable cytotoxicity. The effect is both time and concentration dependent. MeSno does not acutely inhibit synthesis of the enzyme as evidenced by the ability of glucocorticoids or cAMP analogs to induce the enzyme to the same extent in the presence or absence of the drug. The enzyme in extracts of cells treated with the drug is essentially identical with the enzyme from extracts of control cells in terms of thermal stability, immunoprecipitability, and affinities for substrates and cofactor. Addition of MeSno to cell extracts and mixing experiments suggests that the thiopurine does not have any direct effect on enzyme activity. Immunochemical analysis of the rates of synthesis and degradation of the aminotransferase have shown that the enzyme is degraded approximately 3-4 times more rapidly in cells treated with the drug than in control cells. At the same time there is no inhibition of the rate of synthesis of the enzyme.

Insulin as a potent, specific growth factor in a rat hepatoma cell line.

A line or rat hepatoma cells in culture which, in response to serum starvation, become arrested in the early G1 phase of growth, can be stimulated by insulin alone to enter the cell cycle and traverse S phase. A half-maximum response is observed at 30 to 70 picomolar concentrations and the maximum response is essentially identical to that found with optimum serum concentrations.

Comparison of the effects of 6- thio- and 6-methylthiopurine ribonucleoside cyclic monophosphates withtheir corresponding nucleosides on the growth of rat hepatoma cells.

The cyclic nucleotide forms of 6-mercaptopurine and 6-methylmercaptopurine have been found to be cytotoxic to rat hepatoma cells. Studies with an inhibitor of phosphodiesterase suggest that the cytotoxicity of both cyclic nucleotides results principally from conversion to the 5'-nucleotide. A comparison of the two thiopurine cyclic nucleotides with their nucleoside counterparts has suggested that (a) the thio derivatives act by a common mechanism which is different from that exerted by the methylthio derivatives, and (b) the methylthio cyclic nucleotide acts, at least in part, by a mechanism which differs from that exerted by the methylthio nucleoside.