Physiological cyclic AMP stimulation and oncogene mRNA levels in HL-60 cells

Altered gene expression of the proto-oncogenes c-fos and c-myc is associated with differentiation of the human promyelocytic leukemia (HL-60) cells. We studied changes of cyclic AMP levels and c-fos and c-myc mRNA levels after stimulation with theophylline and theophylline plus isoproterenol. Reduced c-fos and c-myc mRNA levels were detected, but the reduction could not, however, be related to the observed alternations in cyclic AMP concentrations. Expression of c-jun and c-Ha-ras was not affected under these conditions.     

Glycogen synthase turnover and phosphorylation in rat H4IIE hepatoma cells

Glycogen synthase was isolated from rat H4IIE hepatoma cells by the use of specific antibodies. Immunoprecipitates from cells grown in the presence of [35S]methionine contained two 35S-labeled polypeptides, designated GS1 and GS2, separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling of both species was half-maximal after 3 h and remained constant up to 48 h. When cells were incubated with [32P]-phosphate, 32P was incorporated into both species with similar kinetics, half-maximal labeling occurring after 2-3 h. The steady-state ratio 32P/35S was significantly higher for the lower mobility GS2 polypeptide. Pulse-chase experiments showed that the two subunits followed similar kinetics with respect to 35S-labeling. However, the turnover of 32P on the GS2 subunit was significantly faster (t1/2 approximately 30 min) than that on the GS1 subunit (t1/2 approximately 2 h). We suggest that the two polypeptides represent different phosphorylation states of the glycogen synthase subunit and are rapidly interconverted.     

Effect of cyclic AMP on RNA and protein synthesis in Candida albicans.

Sequence analysis by the automated Edman degradation shows that dopamine $\text{β}$-hydroxylase (dopamine $\text{β}$-monooxygenase; EC 1.14.17.1) from bovine adrenal medulla contains equal amounts of NH₂-terminal alanine and serine residues. The sequence data are in agreement with the proposal that this enzyme consists of two types of polypeptide chains which are identical in the NH₂-terminal ends, except that one of the chains lack the NH₂-terminal tripeptide Ser-Ala-Thr.     

Oxidative stress induces phosphoenolpyruvate carboxykinase expression in H4IIE cells

Oxidative stress is closely associated with diabetes and is a major cause of insulin resistance. Impairment of hepatic insulin action is thought to be responsible for perturbations in hepatic glucose metabolism. In this study, we found that oxidative stress is involved in the dysregulation of gene expression of phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, by a mechanism independent of insulin. Elevation of oxidative stress by injection of ferric nitrilotriacetate in rats increased the expression of hepatic PEPCK mRNA. To examine the direct action of oxidative stress on PEPCK expression, we treated H4IIE hepatoma cells with buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. BSO increased intracellular oxidative stress and the expression of PEPCK mRNA. Inhibition of p38 mitogen-activated protein kinase (p38 MAP kinase), which mediates responses to oxidative stress, suppressed the induction of PEPCK mRNA by BSO. These results suggest that oxidative stress dysregulates hepatic PEPCK expression by an insulin-independent mechanism.     

Effect of starvation and diabetes on the sensitivity of carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate.

The sensitivity of carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate was decreased markedly in liver mitochondria isolated from either 48 h-starved or streptozotocin-diabetic rats. These treatments of the rat also decreased the sensitivity of fatty acid oxidation by isolated hepatocytes to inhibition by this compound. Furthermore, incubation of hepatocytes prepared from fed rats with N6O2'-dibutyryl cyclic AMP also decreased the sensitivity, whereas incubation of hepatocytes prepared from starved rats with lactate plus pyruvate had the opposite effect on 4-hydroxyphenylglyoxylate inhibition of fatty acid oxidation. The sensitivity of carnitine palmitoyltransferase I of mitochondria to 4-hydroxyphenylglyoxylate increased in a time-dependent manner, as previously reported for malonyl-CoA. Likewise, oleoyl-CoA activated carnitine palmitoyltransferase I in a time-dependent manner and prevented the sensitization by 4-hydroxyphenylglyoxylate. Increased exogenous carnitine caused a moderate increase in fatty acid oxidation by hepatocytes under some conditions and a decreased 4-hydroxyphenylglyoxylate inhibition of fatty acid oxidation at low oleate concentration, without decreasing the difference in 4-hydroxyphenylglyoxylate inhibition between fed- and starved-rat hepatocytes. Time-dependent changes in the conformation of carnitine palmitoyltransferase I or the membrane environment may be involved in differences among nutritional states in 4-hydroxyphenylglyoxylate-sensitivity of carnitine palmitoyltransferase I.     

Effect of hormones on cyclic AMP levels in cultured human cells.

Cultured cells derived from human adipose tissue grew more slowly and had significantly higher basal levels of cyclic AMP than cultured fibroblasts. Cyclic AMP levels in cultured adipose tissue cells were unaffected by epinephrine and were elevated 15-fold by prostaglandin E₁ while fibroblast cyclic AMP levels were elevated 27-fold by epinephrine and 95-fold by prostaglandin E₁. These results support the postulate that the cultured adipose tissue cell is a distinct cell type which may represent an adipocyte or preadipocyte in culture.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP.

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclic AMP which both promote the aggregation of isolated cells into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cyclic AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Osmotic regulation of the heat shock response in H4IIE rat hepatoma cells.

The influence of cell hydration on the heat shock response was investigated in H4IIE hepatoma cells at the levels of HSP70 expression, MAP kinase activation, induction of c-jun and the MAP kinase phosphatase MKP-1, heat resistance, and development of tolerance/sensitization to arsenite after a priming heat treatment. Induction of HSP70, MKP-1, and c-jun by heat was delayed, but more pronounced or sustained, under hyperosmotic conditions compared with normo- and hypo-osmotically exposed cells. Anisosmolarity per se was ineffective to induce HSP70; some expression of the mRNAs for MKP-1 and c-jun in response to hyperosmolarity was found, but was small compared with the response to heat. Heat-induced activation of JNK-1 was increased under hyperosmotic conditions and more sustained than the JNK-activity induced by hyperosmolarity at 37 degrees C. A prominent Erk-2 activation was found immediately after heat shock under hypo- and normo-osmotic conditions, but Erk-2 activation was weak in hyperosmolarity-exposed cells. Despite anisosmotic alterations of the heat shock response at the molecular level, the heat resistance of H4IIE cells toward heat shock was not affected by ambient osmolarity. However, an osmolarity-dependent sensitization to arsenite was induced by a priming heat shock. The osmodependence of the H4IIE cell response to heat differs from that recently found in primary rat hepatocytes. The data are discussed in terms of cellular adaption mechanisms and their physiological relevance.     

Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclin AMP which both promote the aggregation of isolated cell into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells to the acute effect of thyroid-stimulating hormone: only cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cycli AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.     

Glucose synthesis by isolated guinea-pig hepatocytes. Effect of cyclic AMP and dibutyryl cyclic AMP.

In isolated guinea-pig hepatocytes, dibutyryl cyclic AMP stimulated gluconeogenesis from 2 mM galactose by 25 and 40% respectively. In the presence of 0.5 mM theophylline, cyclic AMP (0.1 mM) increased glucose synthesis from lactate and galactose by 26 and 34% respectively.     

Effects of glucagon and insulin on the cyclic AMP binding capacity of hepatocyte cyclic AMP-dependent protein kinase

Extracts obtained from rat hepatocytes incubated with saline, glucagon or insulin were electrophoresed on polyacrylamide gels and then assayed for cyclic (³H)AMP binding capacity. Analysis of the binding patterns demonstrated that glucagon dissociated a holoenzyme of cyclic AMP-dependent protein kinase in a dose-dependent manner. The increase in free regulatory subunits and, hence, in free catalytic subunits explains the activation of this enzyme by glucagon in the liver. Insulin decreased both the amount of cyclic (³H)AMP bound to the holoenzyme and the capacity of the enzyme to be dissociated when the extracts were incubated with increasing concentrations of this cyclic nucleotide. We propose that these insulin-induced effects are determined by an inhibition of the cyclic AMP binding capacity of this protein kinase. This mechanism could account for the inactivation of cyclic AMP-dependent protein kinase that insulin causes in the liver.Abbreviations cAMP (cyclic AMP), Adenosine 3,5 monophosphate - (³H)cAMP cyclic (³H)AMP - MIX 1-methyl-3-isobutylxanthine     

Interdomain interaction of cyclic AMP receptor protein in the absence of cyclic AMP

Interdomain interaction of apo-cyclic AMP receptor protein (apo-CRP) was qualified using its isolated domains. The cAMP-binding domain was prepared by a limited proteolysis, while the DNA-binding domain was constructed as a recombinant protein. Three different regions making interdomain contacts in apo-CRP were identified by a sequence-specific comparison of the HSQC spectra. The results indicated that apo-CRP possesses characteristic modules of interdomain interaction that are properly organized to suppress activity and to sense and transfer the cAMP binding signals. Particularly, the inertness of the DNA-binding motif in apo-CRP was attributable to the participation of F-helices in the interdomain contacts.     

Rapid changes in the concentration of phosphoenolpyruvate carboxykinase mRNA in rat liver and kidney. Effects of insulin and cyclic AMP

Starvation and diabetes both caused a marked increase in the concentration of hepatic phosphoenolpyruvate caroboxykinase mRNA while the administration of insulin to diabetic rats or refeeding glucose to starved animals caused a marked reduction in the levels of enzyme mRNA as measured by hybridization using a cDNA probe.l The Administration of dibutyryl cAMP to a starved-refed cat caused an 8-fold induction of phosphoenolpyruvate carboxykinase mRNA in 1 h. Triamcinolone plus acidosis induced the levels of enzyme mRNA in kidney 3-fold within 6 h, however, starvation for 24h had only marginal effects. In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with the relative levels of translatable mRNA for the enzyme. The half-time of phosphoenolpyruvate carboxykinase mRNA, determined after the administration of either alpha-amanitin or cordycepin to starved animals, was approximately 40 min. However, cycloheximide either alone or together with cordycepin, not only prevented the decrease in phosphoenolpyruvate carboxykinase mRNA sequence abundance, but induced it 2-fold. Cycloheximide itself, when injected into 21-day fetal rats in utero caused an induction of enzyme mRNA equal to that noted when dibutyryl cAMP was administered. The mRNA for phosphoenolpyruvate carboxykinase is approximately 2.8 kb in length, but nuclei from the livers of diabetic rats contain a number of putative precursor RNA species for the enzyme, up to 6.5 kb in size, all containing a poly(A) tail. Two hours after refeedng glucose to a starved rat, these nuclear RNA species could no longer be detected by hybridization to our cDNA probe.     

Osmotic regulation of betaine homocysteine-S-methyltransferase expression in H4IIE rat hepatoma cells

Cell hydration changes critically affect liver metabolism and gene expression. In the course of gene expression studies using nylon cDNA-arrays we found that hyperosmolarity (405 mosmol/l) suppressed the betaine-homocysteine methyltransferase (Bhmt) mRNA expression in H4IIE rat hepatoma cells. This was confirmed by Northern blot and real-time quantitative RT-PCR analysis, which in addition unraveled a pronounced induction of Bhmt mRNA expression by hypoosmotic (205 mosmol/l) swelling. Osmotic regulation of Bhmt mRNA expression was largely paralleled at the levels of Bhmt protein and enzymatic activity. Like hyperosmotic NaCl, hyperosmotic raffinose but not hyperosmotic urea suppressed Bhmt mRNA expression, suggesting that cell shrinkage rather than increased ionic strength or hyperosmolarity per se is the trigger. Hypoosmolarity increased the expression of a reporter gene driven by the entire human BHMT promoter, whereas destabilization of BHMT mRNA was observed under hyperosmotic conditions. Osmosensitivity of Bhmt mRNA expression was impaired by inhibitors of tyrosine kinases and cyclic nucleotide-dependent kinases. The osmotic regulation of BHMT may be part of a cell volume-regulatory response and additionally lead to metabolic alterations that depend on the availability of betaine-derived methyl groups.     

Interaction of insulin,glucocorticoids, and protein kinase C in the regulation of insulin-like growth factor-binding protein-1 production by H4IIE rat hepatoma cells

A sensitive RIA was used to examine regulation of IGFBP-1 in H4IIE rat hepatoma cells. IGFBP-1 was stimulated up to tenfold by dexamethasone and corticosterone, and this stimulation was abolished by RU486. The effect of dexamethasone increased with time in culture. Phorbol 12-myristate 13-acetate (PMA) stimulated IGFBP-1 up to fourfold with a maximal effect in short-term culture. Dexamethasone and PMA were additive in stimulating IGFBP-1. Under basal conditions IGFBP-1 production was linearly related to cell density: however, stimulation by dexamethasone was greatest in confluent cells, and PMA had a greater effect in sparse cultures. Insulin inhibited IGFBP-1 up to 80%, and this effect diminished with time in culture but was unaffected by cell density. Dexamethasone was stimulatory in the presence of a maximal inhibitory concentration of insulin, and insulin was inhibitory in the presence of maximal dexamethasone from 3–48 h in culture, regardless of cell density. PMA abolished the inhibitory action of insulin on IGFBP-1 secretion and mRNA expression during incubation periods of less than 4 h and not during longer incubations. PMA did not influence the stability of IGFBP-1 mRNA. We conclude that, in rat H4IIE cells, dexamethasone and PMA stimulate IGFBP-1 by independent mechanisms and speculate that when protein kinase C is activated the inhibitory action of insulin is blocked. © 1996 Wiley-Liss, Inc.