Insulin-induced gene 33 mRNA expression in Chinese hamster ovary cells is insulin receptor dependent

Gene 33 (g33) is a non-tissue-specific gene regulated in rat liver and hepatoma cells by insulin and other agents. It is thought to participate in the transition from quiescence to proliferation in mitogen-treated cells. The mechanism(s) by which insulin exerts its action on g33 are not totally understood; it is unclear whether a functional insulin receptor is required for this action. In this study, we evaluate the mechanism for insulin induction of g33 mRNA in Chinese hamster ovary (CHO) cells transfected with the neomycin-resistant plasmid (CHONeoB), human insulin receptor (CHONewIRa), and a kinase-defective insulin receptor mutated at the ATP-binding site (CHOK1018A). Transfected cells had higher levels of insulin binding than that of CHONeoB cells; insulin-induced phosphorylation of the insulin receptor and its intracellular substrates were impaired in CHOK1018A cells. Maximal insulin induction of mRNA(g33) occurred 3 h after hormonal exposure in all cell lines. The degree of insulin stimulation of g33 mRNA levels was four- to sixfold higher in CHONewIRa than in CHONeoB or CHOK1018A cells, which had minimal levels of insulin-stimulated g33 mRNA levels. Half-maximal stimulation of g33 mRNA levels was observed at 0.06 +/- 0.01 nM in CHONewIRa cells, consistent with insulin interaction with its own receptor. Wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI3K), had some effects on insulin stimulation of g33 mRNA in CHO NewIRa cells. PD98059, an inhibitor of mitogen-activated kinase kinase (MAPKK), and rapamycin, a p70 S6 kinase inhibitor, had minimal effect on insulin stimulation of g33 mRNA in all cells tested. By contrast, hydroxy-2-naphthalenylmethyl)phosphonic acid triacetoxymethyl ester (HNMPA(AM)(3), a selective inhibitor of the insulin receptor tyrosine kinase, caused complete inhibition of insulin stimulation of g33 mRNA levels. These data indicate that the insulin receptor with intact kinase activity is required for insulin stimulation of g33 mRNA levels. They also suggest that AKT, a PI 3-kinase downstream effector molecule, could mediate insulin stimulation of g33 mRNA. The mechanism(s) of insulin regulation of g33 expression downstream of receptor do not seem to rely entirely on the classic insulin receptor transduction pathway, as a minor effect was observed upon inhibition of MAPKK, suggesting that multiple pathways may be involved.     

Vanadate and molybdate stimulate the oxidation of NADH by superoxide radical

Vanadate or molybdate strongly accelerate the cooxidation of NADH, or of reduced nicotinamide mononucleotide, by the xanthine oxidase plus xanthine reaction. Superoxide dismutase eliminated the effect of vanadate or molybdate, while catalase was without effect. It follows that vanadate or molybdate accelerate the oxidation of dihydropyridines by O-2. A stoichiometry of 4 NADH oxidized per O-2 introduced suggests a chain reaction for which a mechanism is proposed. These results provide an explanation for the reported stimulation, by vanadate, of NADH oxidation by biological membranes.     

Vanadate stimulation of insulin release in normal mouse islets.

The effects of vanadate (Na3VO4) on pancreatic B-cell function were studied in normal mouse islets. Vanadate did not affect basal insulin release but potentiated the effect of 7-30 mM glucose at concentrations of 0.1-1 mM. This effect was progressive and slowly reversible. It was abolished by omission of extracellular Ca2+ but unaffected by blockers of adrenergic or muscarinic receptors. Comparison of the changes in membrane potential, 86Rb efflux and 45Ca efflux that vanadate and ouabain produced in B-cells made it possible to exclude the hypothesis that vanadate increases insulin release by blocking the sodium pump. Vanadate was also without effect on cAMP levels. On the other hand, it markedly changed the characteristics of the Ca(2+)-dependent electrical activity and of the oscillations of cytoplasmic Ca2+ recorded in B-cells stimulated by 15 mM glucose. In the steady state, Ca2+ influx was increased by vanadate, and this resulted in a rise in cytoplasmic Ca2+. The exact mechanisms underlying these changes could not be established but a blockade of K channels was excluded. In the presence of LiCl, vanadate markedly increased inositol phosphate levels in islet cells. This effect was attenuated but not suppressed by omission of Ca2+. A small increase in inositol bisphosphate was still produced by vanadate in the absence of LiCl. These results suggest that vanadate both stimulates phosphoinositide breakdown and inhibits inositol phosphate degradation. In conclusion, vanadate does not induce insulin release, but markedly potentiates the stimulation by glucose. This property is not due to an inhibition of the sodium pump or to a rise in cAMP concentration. It results from a complex interplay between changes in B-cell membrane potential, phosphoinositide metabolism and Ca2+ handling.     

SARs stimulate but do not confer position independent gene expression.

Two minimal scaffold-associated regions (SARs) from Drosophila were tested in stably transformed cells for their effects on the expression of reporter genes. The expression of genes bounded by two SARs is consistently stimulated by about 20- to 40-fold, if the average of a pool of cell transformants is analyzed. However, analysis of individual, stable cell transformants demonstrates that flanking SAR elements do not confer position-independent expression on the reporter gene and that the extent of position-dependent variegation is similarly large with or without the flanking SAR elements. The SAR stimulation of expression is observed in stable but not in transiently transfected cell lines. The Drosophila scs and scs' boundary elements, which do not bind to the nuclear matrix in vitro, are only about one-tenth as active as SARs in stimulating expression in stable transformants. Interestingly, the SAR stimulatory effect can be blocked by a fragment containing CpG islands (approximately 70% GC), if positioned between the SAR and the enhancer. In contrast, when inserted in the same position, control fragments, such as the scs/scs' elements, do not interfere with SAR function.     

Serum,insulin and phorbol esters stimulate rRNA and tRNA gene expression in both dividing and nondividing Drosophila cells

The expression of genes that code for the large ribosomal RNAs (rRNAs) and tRNAs can be regulated by calcium, serum, insulin and a tumor-promoting phorbol ester, TPA. These effectors can rapidly alter rRNA and tRNA synthesis in dividing and nondividing Drosophila cells. In an in vitro assay system of the nondividing cells of the male accessory glands, calcium, insulin and TPA were shown to increase both rRNA and tRNA synthesis. Exposure of actively dividing Drosophila culture cells to differing serum concentrations or TPA also altered rRNA and tRNA synthesis. Nuclear run-on assays demonstrate that the exposure of these cells to increased serum concentrations coordinately alters RNA polymerase I loading on both 18S and 28S rDNA. These data indicate that calcium, growth factors and a tumor-promoter each can signal changes in ribosomal and tRNA gene expression.     

Increase of PTEN gene expression in insulin resistance.

Phosphatase and tensin homologue deleted from chromosome ten (PTEN) has recently been characterized as a regulator of insulin sensitivity in the insulin target tissue. However, whether PTEN gene expression is changed in insulin resistance remains unclear. We observed that both the mRNA and protein level of PTEN in soleus muscle isolated from the obese Zucker rats (Fa/Fa) were increased compared to the age-matched lean group. Similarly, both the mRNA and protein level of PTEN in soleus muscle of the fructose-fed lean Zucker rats (Fa/Fa) showing the higher glucose-insulin index were higher than that of the regular chow fed group. These results suggest that increase of PTEN gene expression seems to be related to the development of insulin resistance.     

Saturated fatty acids stimulate and insulin suppresses CIDE-A expression in bovine mammary epithelial cells

Cell death-inducing DNA fragmentation factor-alpha-like effector A (CIDE-A) was first identified by its sequence homology with the N-terminal domain of DNA fragmentation factor (DFF). CIDE-A negatively regulates the activity of uncoupling protein 1 (UCP1) in brown adipose tissue. CIDE-A and UCP1 mRNA were detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and lactating bovine mammary glands. Physiological concentrations of saturated fatty acids (stearate and palmitate), but not unsaturated fatty acids (oleate and linoleate) induced up-regulation of CIDE-A mRNA in bMEC. Treatment with insulin (5-10 ng/ml) induced down-regulation of CIDE-A and UCP1. The expression levels of CIDE-A and UCP1 mRNA in bovine mammary glands at various stages of the lactation cycle were determined by quantitative RT-PCR analysis. CIDE-A mRNA expression at peak lactation (2 months after parturition) was significantly higher than at dry off and non-pregnancy but not late lactation. These results suggest that CIDE-A and UCP1 are regulated by insulin and/or fatty acids in mammary epithelial cells and lactating mammary glands, and thereby play an important role in lipid and energy metabolism.     

cDNA isolation and gene expression of the maize annexins p33 and p35.

The isolation, cloning, and sequencing of two full-length cDNAs corresponding to the root tip forms of the maize (Zea mays L. cv Clipper) annexins p33 and p35 are described. These are the first complete sequences for the widely reported doublet of plant annexins. The predicted sequences can be divided into four repeat domains characteristic of the annexin family, but Ca2+ binding by the type-II site typical of annexins would be predicted to occur only in repeats 1 and 4. This reduced number of sites is consistent with previously reported biochemical data indicating a high Ca2+ requirement for membrane association. Although the two annexins are very similar (80% amino acid identity), their genes are quite distinct, as demonstrated by their different 3' noncoding regions and Southern blotting. The predicted sequences of the root tip proteins are very similar to regions known from peptide sequencing of the coleoptile proteins. Because a rather small gene family is indicated, the implication is that there may be less functional diversity than in animal cells. Furthermore, the sequence data clearly show that plant annexins form a very distinct group compared with those from other kingdoms.