Constitutively active phosphatidylinositol 3-kinase and AKT are sufficient to stimulate the epithelial Na+/H+ exchanger 3.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors and has been shown to be involved in regulation of stimulated exocytosis and endocytosis. One of the downstream signaling molecules activated by PI 3-kinase is the protein kinase Akt. Previous studies have indicated that PI 3-kinase is necessary for basal Na(+)/H(+) exchanger 3 (NHE3) transport and for fibroblast growth factor-stimulated NHE3 activity in PS120 fibroblasts. However, it is not known whether activation of PI 3-kinase is sufficient to stimulate NHE3 activity or whether Akt is involved in this PI 3-kinase effect. We used an adenoviral infection system to test the possibility that activation of PI 3-kinase or Akt alone is sufficient to stimulate NHE3 activity. This hypothesis was investigated in PS120 fibroblasts stably expressing NHE3 after somatic gene transfer using a replication-deficient recombinant adenovirus containing constitutively active catalytic subunit of PI 3-kinase or constitutively active Akt. The adenovirus construct used was engineered with an upstream ecdysone promoter to allow time-regulated expression. Adenoviral infection was nearly 100% at 48 h after infection. Forty-eight hours after infection (24 h after activation of the ecdysone promoter), PI 3-kinase and Akt amount and activity were increased. Increases in both PI 3-kinase activity and Akt activity stimulated NHE3 transport. In addition, a membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and increases PI 3-kinase activity similarly enhanced NHE3 transport activity and also increased the percentage of NHE3 on the plasma membrane. The magnitudes of stimulation of NHE3 by constitutively active PI 3-kinase, PI 3-kinase peptide, and constitutively active Akt were similar to each other. These results demonstrate that activation of PI 3-kinase or Akt is sufficient to stimulate NHE3 transport activity in PS120/NHE3 cells.

Cysteine 981 of the human insulin receptor is required for covalent cross-linking between beta-subunit and a thiol-reactive membrane-associated protein.

The cytoplasmic domain of the insulin receptor (IR) beta-subunit contains cysteine (Cys) residues whose reactivity and function remain uncertain. In this study, we examined the ability of the bifunctional cross-linking reagent 1,6-bismaleimidohexane (BMH) to covalently link IR with interacting proteins that possess reactive thiols. Transfected Chinese hamster ovary cells expressing either the wild-type human IR, C-terminally truncated receptors, or mutant receptors with Cys --> Ala substitutions and mouse 3T3-L1 adipocytes were used to compare the BMH effect. The results showed the formation of a large complex between the wild-type human receptor beta-subunit and molecule X, a thiol-reactive membrane-associated protein, in both intact and semipermeabilized cells in response to BMH. Prior cell stimulation with insulin had only a modest effect in this process. Western blot analysis revealed that the receptor alpha-subunit was not present in the beta-X complex. The BMH cross-linking did not inhibit in vitro tyrosine phosphorylation of the receptor complexed with molecule X. Both the human IR Cys981Ala mutant and murine IR, that lacks the equivalent of human Cys(981), failed to react with BMH. Finally, no covalent association between IR beta-subunit and IRS-1, the protein tyrosine phosphatase LAR or SHP-2 was observed in BMH-treated cells expressing the wild-type human IR. These results demonstrate a striking difference in reactivity among the cytoplasmic IR beta-subunit thiols and clearly show that Cys(981) of human IR beta-subunit is in close proximity to a thiol-reactive membrane-associated protein under basal and insulin-stimulated conditions.

Modulation of CCAAT/enhancer-binding protein-alpha gene expression by metabolic signals in rodent adipocytes.

The transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) is a positive modulator of transcription for several adipocyte-specific genes that play a role in energy metabolism. However, there is little information available regarding the regulation of its expression by metabolic signals. Exposure to insulin for 5-24 h attenuated C/EBPalpha expression when 3T3-L1 adipocytes were incubated in 24 mM glucose, but not in 5.7 mM glucose. Nuclear run-on transcription assays indicated a transcriptional repression of C/EBPalpha gene, but not that of C/EBPbeta. Glucosamine, a product of the hexosamine pathway, in the presence of low glucose mimicked high glucose's ability to reduce C/EBPalpha messenger RNA expression in insulin-treated cells. Similar results were obtained with xylitol, an activator of the pentose phosphate pathway. There was no correlation between the accumulation of hexosamine pathway metabolites (e.g. UDP-N-acetylhexosamines) and/or changes in intracellular protein glycosylation with the ability of high glucose, glucosamine, or xylitol to down-regulate C/EBPalpha gene expression. None of these treatments caused a reduction in intracellular ATP levels. Stable transfection of 3T3-L1 cells with the 5'-flanking 468-bp sequence of the mouse C/EBPalpha gene fused to luciferase demonstrated that promoter activity was also reduced by these nutrients. Of interest, treatment of rats with glucose or glucosamine led to a reduction in C/EBPalpha messenger RNA levels in epididymal, but not omental, fat. Taken together, these results suggest that metabolic signals serve to down-regulate C/EBPalpha expression both in vitro and in vivo.

Antiapoptotic signaling by the insulin receptor in Chinese hamster ovary cells.

We have sought to determine whether insulin can promote cell survival and protect Chinese hamster ovary (CHO) cells from apoptosis induced by serum starvation. Low concentrations of insulin were antiapoptotic for cells overexpressing wild-type insulin receptors but not in cells transfected with kinase-defective insulin receptor mutants that lacked a functional ATP binding site. However, treatment with orthovanadate (50 microM), a widely used tyrosine phosphatase inhibitor, led a dramatic reduction in internucleosomal DNA fragmentation in both cell lines. Cells transfected with truncated receptor mutants in either the juxtamembrane or C-terminal domain were as responsive as cells overexpressing wild-type receptors in mediating insulin antiapoptotic protection. The mechanisms underlying insulin antiapoptotic protection were investigated using a variety of pharmacological tools known to inhibit distinct signaling pathways. The phosphatidylinositol-3' kinase inhibitors wortmannin and LY294002 had only a modest influence whereas blocking protein farnesylation with manumycin severely disrupted the antiapoptotic capacity of the insulin receptor. Of interest, cells gained antiapoptotic potential following inhibition of extracellular signal-regulated kinase activation with the pharmacological agent PD98059. Insulin induced MKK3/MKK6 phosphorylation and activation of p38 MAP kinase whose activity was inhibited with SB203580. However, the inhibition of p38 MAP kinase had no effect on the protection offered by insulin. We conclude that the antiapoptotic function of the insulin receptor requires intact receptor kinase activity and implicates a farnesylation-dependent pathway. Increase in cellular phosphotyrosine content, however, triggers antiapoptotic signal that may converge downstream of the insulin receptor.

Nucleotide excision repair is not required for the antiapoptotic function of insulin-like growth factor 1.

The expression of ERCC1, a member of the nucleotide excision repair (NER) family, is enhanced in cells transfected with insulin-like growth factor 1 (IGF-1) receptors. Of interest, an excellent concordance between ERCC1 expression and NER-mediated cell survival has been demonstrated. The two aims of the present study were to determine the signaling pathways used by IGF-1 to confer protection against apoptotic cell death in Chinese hamster ovary (CHO) cells and to assess the role of NER in this IGF-1 action. Experiments with pharmacological inhibitors indicated that phosphatidylinositol 3-kinase (PI 3-kinase) but not mitogen-activated protein kinase (ERK1/ERK2) mediates IGF-1 antiapoptotic activity. Using two series of CHO cells that have altered expression of ERCC1 or XPB/ERCC3, we examined IGF-1's ability to delay apoptotic death and reduction of mitochondrial oxidative function mediated by growth factor withdrawal. IGF-1 effectively blocked apoptosis, concomitant with increased MTT activity, in a pair of CHO cell lines expressing inactive ERCC1 (43-3B cells) and the transfected line of the mutant carrying the expressed human ERCC1 gene (83-G5 cells). Similarly, repair-deficient UV24 cells, which lack XPB/ERCC3, and their parental line AA8 were also responsive to the IGF-1's antiapoptotic capacity. In the presence of IGF-1, these cell lines became resistant to the cleavage of poly(ADP-ribose) polymerase, a key player in DNA damage recognition and DNA repair. These results suggest that PI 3-kinase activation plays a determinant role in the antiapoptotic function of IGF-1, but that functional NER does not play a critical part in mediating this IGF-1 response.

Involvement of the Ras/extracellular signal-regulated kinase signalling pathway in the regulation of ERCC-1 mRNA levels by insulin.

Expression of DNA repair enzymes, which includes ERCC-1, might be under the control of hormonal and growth factor stimulation. In the present study it was observed that insulin increased ERCC-1 mRNA levels both in Chinese hamster ovary cells overexpressing human insulin receptors (HIRc cells) and in fully differentiated 3T3-L1 adipocytes. To investigate the mechanisms underlying the increase in ERCC-1 gene expression in HIRc cells, we used a variety of pharmacological tools known to inhibit distinct signalling pathways. None of these inhibitors affected the amount of ERCC-1 mRNA in unstimulated cells. The pretreatment of cells with two chemically unrelated phosphatidylinositol 3'-kinase inhibitors, wortmannin and LY294002, failed to block the doubling of ERCC-1 mRNA content by insulin. Similarly, inhibition of pp70 S6 kinase by rapamycin had no apparent effects on this insulin response. In contrast, altering the p21(ras)-dependent pathway with either manumycin, an inhibitor of Ras farnesylation, or PD98059, an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase, suppressed the induction of ERCC-1 mRNA by insulin (P<0.001). Furthermore inhibition of RNA and protein synthesis negatively regulated the expression of this insulin-regulated gene (P<0.005). These results suggest that insulin enhances ERCC-1 mRNA levels by the activation of the Ras-ERK-dependent pathway without the involvement of the phosphatidylinositol 3'-kinase/pp70 S6 kinase.

Overexpression and activation of the insulin receptor enhances expression of ERCC-1 messenger ribonucleic acid in cultured cells.

In this study, a partial hamster complementary DNA encoding ERCC-1, a member of the DNA excision repair gene family, has been cloned. The nucleic acid and amino acid sequences were highly homologous to those of human and mouse ERCC-1. The hamster ERCC-1 gene was expressed as a 1.2-kilobase message in cultured Chinese hamster ovary cells. Northern (RNA) blot analysis revealed that overexpression of the insulin receptor or various growth factor receptor tyrosine kinases in Chinese hamster ovary cells increased ERCC-1 messenger RNA (mRNA) levels. This effect did not occur in cells overexpressing mutated insulin receptors that are known to have impaired kinase-related signaling. Increased ERCC-1 expression correlated with resistance to UV exposure. Fluorescent-activated cell sorter analysis of confluent cell populations indicated no differences in cell cycle distribution. Furthermore, no significant relationship was demonstrated between the relative expression of ERCC-1 mRNA and the rate of glucose utilization. Insulin enhanced the accumulation of ERCC-1 mRNA in serum-deprived cells expressing wild-type insulin receptors. The potential role for activation of the insulin receptor and related growth factor receptors in ERCC-1 gene expression and function remains to be defined.

Selection of DNA clones with enhancer sequences.

A method is described for selection of DNA clones that contain enhancer sequences that activate gene expression. An Escherichia coli-rodent cell shuttle vector, pPyE0, was used that contains polyoma viral DNA without the polyoma enhancer region. Replication of pPyE0 DNA in mouse cells is markedly reduced due to deletion of the polyoma enhancer region. Insertion of mouse genomic DNA fragments that contain putative enhancer sequences into pPyE0 adjacent to the polyoma origin of replication restored, to varying extents, the ability of the recombinant plasmid DNA to replicate in mouse cells. Recombinant plasmids that replicate well in mouse cells, therefore, are amplified selectively. Transfection of mouse neuroblastoma or fibroblast cells that constitutively synthesize polyoma large tumor antigen with a library of mouse genomic DNA fragments inserted in pPyE0 yielded many recombinant plasmids. DNA inserts from each of the 16 clones that were examined stimulated the expression of an enhancerless chloramphenicol acetyltransferase reporter gene. The DNA inserts from 4 clones that were studied resulted in 4- to 13-fold increases in chloramphenicol acetyltransferase mRNA in transfected mouse cells. Nucleotide sequence analysis led to the identification of 5 genomic DNA clones that were obtained by selection. All of the homologies found were to regions of DNA that are thought to be involved in the regulation of gene expression.

Effect of angiotensin II on vasopressin in plasma and platelets in SH and WKY rats.

A study was carried out of the effects of iv angiotensin II on vasopressin release and the distribution of vasopressin between platelets and plasma in 12 week old conscious unrestrained SH and WKY rats. Angiotensin II was infused at rates of 31.25 to 500 ng/kg X min for 20 min. There was an enhanced pressor responsiveness to angiotensin II in the SH rats. Angiotensin II caused a moderate increase in plasma vasopressin concentrations, but only at doses which produced maximal pressor responses (250 to 500 ng/kg X min). There were no significant differences in vasopressin responses to angiotensin II in SH compared to WKY rats. Approximately 30% of circulating immunoreactive vasopressin was found in platelets in both SH and WKY rats, and this distribution was not greatly affected by the iv infusion of angiotensin II.

One-clip, one-kidney hypertension in rats with hereditary hypothalamic diabetes insipidus.

An attempt was made to produce one-clip, one-kidney hypertension in the rat with diabetes insipidus (DI). Renal artery constriction in unilaterally nephrectomized DI rats (DI-clip) resulted in an increased blood pressure in all 9 rats, but this response was only transient in 3 rats. The magnitude of the hypertension was less in the DI-clip rats than in Long-Evans rats subjected to the same protocol (LE-clip). Infusion of saralasin i.v. at doses of 10 and 30 micrograms/kg . min. 4 to 6 weeks after surgery was without effect on mean arterial pressure in LE-clip and control DI rats, but substantially lowered blood pressure in the DI-clip rats (p less that 0.05 - 0.01). It is concluded that vasopressin is not essential for the production of one-clip, one kidney hypertension in the rat, and that, in the DI rat, the renin-angiotensin system is an important factor in this form of hypertension.

Vasopressin in the rat with partial nephrectomy-salt hypertension.

The role of vasopressin in the pathogenesis of partial nephrectomy (PN)-salt hypertension was examined in the rat. Hypertension was produced by reducing renal mass 70% and substituting 1% saline for drinking water 2 to 4 days after surgery. PN alone resulted in an increase in systolic blood pressure. Subsequent salt loading led to a further large increase in arterial pressure. On the second to third day after substitution of saline for drinking water, urinary vasopressin excretion (UADHV) was increased six-fold and the plasma vasopressin concentration was increased two and one-half-fold. UADHV then fell to a level that was three-fold greater than control values 5 days later. Although there was a marked stimulation of vasopressin release during the period of salt loading, a vasopressin pressor antagonist had only a small effect on arterial pressure. This suggests vasopressin is not a major pressor agent in PN-salt hypertension.

The importance of vasopressin in the development and maintenance of DOC-salt hypertension in the rat.

Experiments were performed to determine the role of vasopressin in deoxycorticosterone (DOC)-salt hypertension. In order to determine if vasopressin is necessary for the development of DOC-salt hypertension, rats with hereditary diabetes insipidus (DI) and normal Long-Evans rats (LE) were unilaterally nephrectomized, treated with DOC Pivalate (30 mg/kg . week) and given saline to drink for 8 weeks. A second group of DI rats were unilaterally nephrectomized, but received no treatment. Systolic blood pressure (SBP) increased 40 mm Hg in the LE group (p less than 0.01) but failed to increase significantly in either DI group. Urinary excretion of vasopressin (UADHV) and SBP were measured in unilaterally nephrectomized LE rats treated with DOC and salt (DOC-LE), salt alone (NaCl-LE) and untreated rats (H2O-LE). The UADHV was elevated in DOC-LE (p less than 0.01) and NaCl-LE (p less than 0.05), but only the DOC-LE rats became hypertensive. Finally, the I.V. injection of analogs of vasopressin, which block its pressor but not antidiuretic activity, lowered mean arterial blood pressure 27 +/- 5 mm Hg in 11 conscious DOC-salt hypertensive rats. It is concluded that vasopressin plays a major role as a pressor agent in both the onset and maintenance of DOC-salt hypertension.