Increase in translatable mRNA for mitochondrial pyruvate carboxylase during differentiation of 3T3 preadipocytes

During differentiation of 3T3 preadipocytes into adipocytes the activity of pyruvate carboxylase, a key lipogenic enzyme, rises about 20-fold. This increase of enzymatic activity is correlated with a comparable rise in the rate of incorporation of [³⁵S]methionine into immunoadsorbable pyruvate carboxylase. Polyadenylated RNA, isolated from differentiated 3T3 adipocytes, directs the synthesis of pyruvate carboxylase in a messenger-dependent reticulocyte lysate translation system at a 18-fold greater rate than that isolated from undifferentiated cells. Thus, it appears that the differentiation-induced rise in the cellular level of pyruvate carboxylase results from an increased rate of carboxylase synthesis due to a rise in the level of translatable carboxylase messenger RNA.     

Ribonucleic acid synthesis in the renal cortex at the initiation of compensatory growth.

The mechanisms responsible for the increase in RNA per cell during the first 48h of renal compensatory growth were studied in the renal cortex. Unilaterally nephrectomized, sham-operated or non-operated rats were used. Incorporation into RNA of labelled precursors was studied in vivo and in vitro. Sham-operation produced significant changes in precursor incorporation, absolute amounts of UTP and RNA, and the rate of RNA synthesis. At 6h after surgery, the amount of RNA decreased in sham-operated controls, whereas that in growing cortex remained unchanged. Incorporation into RNA in vivo was greater in the growing cortex, although the rate of RNA synthesis was not increased. At 24h, precursor incorporation into RNA and UTP and RNA synthesis were all increased in the growing cortex. In contrast with results obtained in vivo, slices of growing cortex incorporated less labelled precursor into RNA than did cortex slices from sham-operated controls, from 3 to 48h. Maximal differences were found from 6 to 24h. An attempt was made to equalize endogenous precursor pool sizes by increasing the concentration of unlabelled uridine in the media; incorporation differences were narrowed significantly. Serum from nephrectomized animals did not increase precursor incorporation into RNA in vitro. An increase in RNA synthesis is an important factor in RNA accretion in the renal cortex beyond 12h of compensatory growth. This is accompanied by increased UTP content and preceded by expansion of other pools. The amount of labelled precursor incorporated into RNA is greatly influenced by its delivery rate to the growing kidney in vivo and by intracellular dilution of expanded precursor pools in vitro.     

TR4 promotes fatty acid synthesis in 3T3-L1 adipocytes by activation of pyruvate carboxylase expression

We show that testicular orphan nuclear receptor 4 (TR4) increases the expression of pyruvate carboxylase (PC) gene in 3T3-L1 adipocytes by direct binding to a TR4 responsive element in the murine PC promoter. While TR4 overexpression increased PC activity, oxaloacetate (OAA) and glycerol levels with enhanced incorporation of ¹⁴C from ¹⁴C-pyruvate into fatty acids in 3T3-L1 adipocytes, PC knockdown by short interfering RNA (siRNA) or inhibition of PC activity by phenylacetic acid (PAA) abolished TR4-enhanced fatty acid synthesis. Moreover, TR4 microRNA reduced PC expression with decreased fatty acid synthesis in 3T3-L1 adipocytes, suggesting that TR4-mediated enhancement of fatty acid synthesis in adipocytes requires increased expression of PC gene.     

Thyroid hormones and muscle protein turnover. The effect of thyroid-hormone deficiency and replacement in thryoidectomized and hypophysectomized rats.

We have investigated the effects of thyroidectomy, hypophysectomy and 3,3',5-tri-iodothyronine replacement on protein synthesis and degradation in skeletal muscle in vivo. Thyroidectomy resulted in a decrease in the rate of protein synthesis as a result of a loss of RNA. However, RNA activity, the rate of protein synthesis per unit of RNA, was not decreased. This was the case in both young growing rats and mature nongrowing rats. Tri-iodothyronine treatment of thyroidectomized rats increased protein synthesis by increasing RNA concentration without changes in RNA activity, and this occurred even when food intake was restricted to prevent any increase in growth. The rate of protein degradation was decreased by thyroidectomy and increased by tri-iodo-thyronine replacement in both animals fed ad libitum and food-restricted animals. Hypophysectomy decreased protein synthesis by decreasing both RNA concentration and activity. these changes were reversed by tri-iodothyronine treatment even in the presence of persistent marked hypoinsulinaemia. This indicates that tri-iodothyronine can activate athe translational phase of protein synthesis in muscle in the absence of significant quantities of insulin. However, tri-iodothyronine does not seem to be obligatory for the maintenance of normal RNA activity in muscle, since in the thyroidectomized rat, in which plasma insulin concentrations are normal, RNA activity is maintained. From a consideration of the magnitude of changes in RNA activity observed in these experiments, it would appear that alterations in rates of elongation as well as initiation are involved in the changes in RNA activity.     

Kinetics of the synthesis of nucleic acids in haploid and diploid loach embryos]

Al kinetic analysis of incorporation of the mixture of 3H-nucleosides in the nucleic acid fractions was carried out to examine the mechanisms of compensation of the genetic material deficiency. Both the haploid and diploid embryos of the loach (Misgurnus fossilis L.) were analyzed. When comparing the DNA and RNA syntheses, the level of phosphorylation (nucleotide pool) in the both genetic variants was under control. The rate of incorporation of the labelled nucleosides in DNA was shown to be higher in haploids at the early developmental stages than in diploids but later it became the same. The increased level of DNA replication in the early haploid embryos was due to the compensatory increase of the cell number in them as compared with the diploid ones. The rate of total RNA synthesis corrected by the differences in the rate of nucleoside phosphorylation varied directly with the degree of ploidy at the blastula stage; at the gastrula stage the value of RNA synthesis per haploid genome was compensated, and at the stage of organogenesis the production of total RNA, as calculated per cell, became in haploids even higher than in diploids. The data obtained suggest the essential changes in the patterns of RNA synthesis control during development.     

Some aspects of metabolic adaptations in lipid metabolism during starvation are mimicked by epinephrine in rat adipocytes

1. The effects of fasting on the neutral lipid synthesis to insulin and/or epinephrine in isolated fat cells have been examined using [1-14C]glucose. 2. The ability of adipocytes from starved rats to synthesize fatty acids from both labeled substrates was markedly diminished compared to adipocytes from control rats. 3. The response of lipogenic stimulation to insulin at all concentrations tested was greatly diminished in adipocytes from 24 hr starved rats. 4. [1-14C]glucose utilization rates in the absence or in the presence of insulin were not significantly different in adipocytes from 24 hr starved rats as compared with control adipocytes, although basal and insulin stimulated glyceride-glycerol synthesis were significantly higher in starved adipocytes. 5. Epinephrine acutely inhibited [1-14C]acetate incorporation into fatty acids for insulin-stimulated lipogenesis in control adipocytes, in contrast, this lipolytic agent strongly increased [1-14C]glucose conversion to triacylglycerols. 6. In both cases, the differences in lipid synthesis capacities found in both nutritional states were abolished by epinephrine.     

Macromolecular synthesis during plant embryogeny: rates of RNA synthesis in Phaseolus coccineus embryos and suspensors

A comparative study of RNA metabolism as an indicator of major changes of tissue organization, cell number, and physiology in the two developmentally and cytologically distinct parts of the bean embryo, the organogenetic part and the suspensor, was carried out. The metabolism of RNA was determined separately for these two parts of embryos removed aseptically from seeds at different times during embryogeny and incubated in culture medium containing ³H-adenosine. Equilibration of ATP in the nucleotide pool, ATP pool size and specific activity, total RNA content, rate of RNA synthesis in culture, rate of RNA synthesis and specific activity during embryogeny, and total protein content were determined. Synthetic activity of the suspensor was highest early in development and then declined, whereas synthetic activity of the organogenetic part increased throughout development. These changes may reflect developmental and functional differences in the two parts of the embryo.     

Adipose tissue metabolism during early pregnancy in the rat: temporal relationships of changes in the metabolic activity, number of insulin receptors, and serum hormone concentrations

The temporal relationships between changes in rates of fatty acid and acylglycerol glycerol synthesis; the activity of lipoprotein lipase of parametrial adipocytes and their capacity to bind insulin; and the serum concentrations of insulin, progesterone, prolactin, and total lactogenic activity have been examined in rats during the first 15 days of pregnancy. The rate of fatty acid and acylglycerol synthesis showed a transient increase at Days 9 and 12 of pregnancy, whereas there was no change in the activity of lipoprotein lipase activity except for a fall between Days 12 and 15 of pregnancy. The capacity of adipocytes to bind insulin was increased by Day 6 of pregnancy and remained elevated until at least Day 15; no changes in the affinity for insulin were observed. Serum progesterone, insulin, and total lactogenic activity were elevated by Days 3, 9, and 12 of pregnancy, respectively. The results show that progesterone but not placental lactogen could be responsible for the rise in the insulin-binding capacity of rat adipocytes during pregnancy, whereas the fall in lipogenic capacity at about Day 12 of pregnancy coincides with the rise in serum placental lactogen.     

Temperature dependence of RNA synthesis parameters in Escherichia coli

For Escherichia coli B/r growing in glucose minimal medium, the following parameters of RNA synthesis remained invariant between 20 and 40 degrees C: RNA polymerase concentration (RNA polymerase/mass), rRNA and tRNA concentration (RNA/mass), RNA polymerase activity (fraction of total RNA polymerase actively engaged in RNA chain elongation), and stable RNA synthesis relative to total RNA synthesis. The following parameters increased 3.4-fold over the same temperature range: rRNA chain elongation rate, guanosine tetraphosphate (ppGpp) concentration, and culture growth rate. Above 40 degrees C, the changes became more complex, and the growth rate began to decrease. The observation that most RNA synthesis parameters are temperature invariant despite the increase of ppGpp suggests that the mechanism of RNA synthesis control by ppGpp, assumed to involve an interaction of RNA polymerase wtih ppGpp, is itself temperature dependent such that, with increasing temperature, higher concentrations of ppGpp are required to affect the RNA polymerase.     

Insulin regulation of protein biosynthesis in differentiated 3T3 adipocytes. Regulation of glyceraldehyde-3-phosphate dehydrogenase

The effect of insulin on protein biosynthesis was examined in differentiated 3T3-L1 and 3T3-F442A adipocytes. Insulin altered the relative rate of synthesis of specific proteins independent of its ability to hasten conversion of the fibroblast (preadipocyte) phenotype to the adipocyte phenotype. Although more than one pattern of response to insulin was observed, we focused on the induction of a Mr 33,000 protein which was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Exposure of 3T3 adipocytes to insulin throughout differentiation specifically increased GAPDH activity and protein content by 2- to 3-fold as compared to 3T3 adipocytes differentiated in the absence of insulin. These changes in enzyme activity and content could be accounted for by a 4-fold increase in the relative rate of synthesis of GAPDH and a 9-fold increase in hybridizable mRNA levels. Within 2 h of insulin addition to 3T3 adipocytes differentiated in the absence of hormone, hybridizable GAPDH mRNA levels increased 3-fold, and within 24 h GAPDH mRNA levels increased 8-fold, and [35S] methionine incorporation into GAPDH protein increased 5-fold. The increase in GAPDH mRNA and GAPDH biosynthesis could be demonstrated using physiologic concentrations of insulin (0.24 nM), indicating that these effects are mediated through a specific interaction with the insulin receptor. These studies demonstrate that insulin, as the sole hormonal perturbant, can increase the synthesis of certain 3T3 adipocyte proteins by altering the cellular content of a specific mRNA.     

Role of A kinase anchor proteins in the tissue-specific regulation of lipoprotein lipase

Activation of protein kinase A by catecholamines inhibits lipoprotein lipase (LPL) activity through the elaboration of an RNA binding complex, which inhibits LPL translation by binding to the 3'-untranslated region of the LPL mRNA. To better define this process, we reconstituted the inhibitory RNA binding complex in vitro and demonstrated that the K homology (KH) domain of A kinase anchor protein (AKAP) 121/149 plays a vital role in the inhibition of LPL translation. Inhibition of LPL translation occurred in vitro only when the Calpha subunit, R subunit, and AKAP 149 were present. Using different glutathione-S-transferase fusion proteins of AKAP 149, sequences containing the KH domain were required for inhibition of LPL translation, and the inhibition of AKAP 121 expression in 3T3-F442A adipocytes with short interfering RNA resulted in loss of epinephrine-mediated translation inhibition. After epinephrine injection into mice, LPL activity was inhibited in white adipose tissue but not in brown adipose tissue (BAT) or muscle. LPL activity and synthetic rate were inhibited in vitro by the addition of epinephrine to 3T3-F442A adipocytes, but there was no effect in L6 muscle cells and cultures of brown adipocytes. Corresponding with these differences in LPL translation, AKAP 121 protein and mRNA were abundantly expressed in mouse white adipose tissue, but was either very low or undetectable in BAT and muscle. Thus, AKAP 121/149 contains a KH region that is essential to the translation inhibition of LPL in response to epinephrine. BAT and muscle do not express significant AKAP 121/149, and this likely explains some of the tissue-specific differences in LPL regulation.     

Insulin-stimulated protein synthesis in adipocytes. Enhanced rate of initiation associated with increased phosphorylation of ribosomal protein S6

The mechanisms of insulin stimulation of protein synthesis in adipocytes are presently unknown. Addition of 10 nM insulin to isolated rat adipocytes caused a 1.5-2.5-fold increase in the protein synthetic rate and a corresponding increase in nascent chain level, indicating that the effect of insulin on protein synthesis in adipocytes is mediated by a stimulation of ribosomal initiation. The effect on protein synthesis exhibited a lag time of 6-8 min after insulin addition. A similar time dependence was also observed for the insulin-induced phosphorylation of ribosomal protein S6. This supports the proposal that these two phenomena are causally linked.     

Changes in RNA metabolism and accumulation of presumptive messenger RNA during transition from the growing to the quiescent state of cultured mouse fibroblasts.

Mouse fibroblasts maintained in tissue culture regulate total protein and ribosomal RNA synthesis co-ordinately with changes in the cellular growth state. Here we show that changes in the rate of synthesis of nuclear non-polyadenylic acid-containing RNA and the rate of accumulation and breakdown of cytoplasmic ribosomal RNA also accompany the transition from the resting to the growing cellular growth state, while the rate of synthesis of nuclear poly (A)-containing RNA and the rates of accumulation and breakdown of cytoplasmic poly(A) containing RNA (presumptive messenger RNA) are, however, only marginally changed. The small net increase (20% to 30%) in the amount of presumptive mRNA is considerably less than the observed increase in protein synthesis (two to threefold) during this transition. We also isolated and characterized extra-polysomal poly(A)-containing ribonucleoprotein particles from quiescent cultures that were similar to those particles obtained by treatment of polyribosomes with EDTA. These experiments suggest that the early increase in protein synthetic activity when quiescent, cultured cells are induced to grow is partially caused by an increased attachment of pre-existing mRNA molecules to free ribosomes.     

Selective inhibition of precursor incorporation into ribosomal RNA in gamma-irradiated Tetrahymena pyriformis.

Sublethal doses of gamma radiation are known to inhibit total RNA synthesis in the ciliate protozoan Tetrahymena. To determine if the synthesis of a particular class of RNA is preferentially inhibited, pulse-labeled RNA was isolated from normal exponentially growing cells, irradiated cells, and cells in which total RNA synthesis had recovered to the pre-irradiation level. The RNAs were analyzed by SDS-polyacrylamide gel electrophoresis and oligo(dT)-cellulose column chromatography. Inhibition of RNA synthesis primarily involves ribosomal RNA. However, radiation does not cause a delay in the processing of precursor rRNA or a preferential loss of either of the mature rRNAs. Following irradiation, poly(A)-containing RNA [poly(A+)RNA] is synthesized at a rate up to three times greater than the control rate. The elevated poly(A+)RNA synthesis occurs during the period of depressed rRNA synthesis and even after rRNA synthesis has recovered to its pre-irradiation rate. While the sizes of the total cellular ribonucleoside triphosphate pools are depressed in the irradiated cells, these pools probably do not represent the actual compartments containing the precursors for RNA synthesis, and the observed changes cannot explain the modifications in macromolecular synthesis in irradiated Tetrahymena.     

Changes in RNA Synthesis and Messenger RNA Content in the Cerebellum of Rats with Graft Versus Host Disease

Cerebellar RNA accumulation, synthesis, and functional capacity was studied in 14-day-old F1 hybrid rats subjected to neonatally induced graft versus host disease (GVHD). There was a decrease in RNA synthetic rate as measured by the uptake of labeled precursors into RNA. The decrease in total cerebellar RNA synthesis was reflected both in a reduced amount of Nissl substance, visible in cresyl violet-stained 10-micron-thick sections of cerebella, and in the total amount of cytoplasmic RNA isolated from individual cerebella from diseased animals compared with control littermates. Analysis of the RNA translational capacity in wheat germ protein synthesizing systems showed that RNA from experimental animals was also biologically less active. Qualitative differences between protein populations in control and diseased animals were analyzed by two-dimensional gel electrophoresis. There were few alterations in the steady state levels of cerebellar protein. However, two-dimensional gel electrophoresis of the peptides synthesized in vitro by RNA from control and diseased animals showed that there were several changes in the relative abundance of some mRNAs between the two RNA populations. These data show that the cerebellar RNA from rats with GVHD differs both qualitatively and quantitatively from that of controls.     

Effect of salt and water stress on RNA synthesis in the excised embryo axis of germinating mung bean (Phaseolus aureus ROXB).

The rate of synthesis of RNA was studied under salt and water stresses created by the addition of four different salts and polyethylene glycol-6000 (PEG-6000) in excised embryo axis of mung bean using^l4C-uracil. Iso-osmotio concentrations of salts and PEG resulted in differential effect on RNA synthesis. PEG inhibited, whereas salt stimulated incorporation of labelled uracil into RNA. Ionic differences on RNA synthesis were very clear as synthesis of RNA was more affected with sulphate than chloride and with sodium than potassium ions.     

Short-lived minus-strand polymerase for Semliki Forest virus

Semliki Forest virus (SFV)-infected BHK-21, Vero, and HeLa cells incorporated [3H]uridine into 42S and 26S plus-strand RNA and into viral minus-strand RNA (complementary to the 42S virion RNA) early in the infectious cycle. Between 3 and 4 h postinfection, the synthesis of minus-strand RNA ceased in these cultures, although the synthesis of plus-strand RNA continued at a maximal rate. At the time of cessation of minus-strand RNA synthesis, two changes in the pattern of viral protein synthesis were detected: a decrease in the translation of nonstructural proteins and an increase in the translation of the viral structural proteins. Addition of cycloheximide and puromycin to cultures of SFV-infected BHK cells actively synthesizing both viral plus- and minus-strand RNA resulted within 15 to 30 min in the selective shutoff of minus-strand RNA synthesis. Removal of the cycloheximide-containing medium led to the resumption of minus-strand synthesis and to an increased rate of viral RNA synthesis. We conclude that the minus-strand polymerase regulates the rate of SFV plus-strand RNA synthesis by determining the number of minus-strand templates and that the synthesis of the minus-strand templates is regulated at the level of translation by a mechanism which utilizes one or more short-lived polymerase proteins.