Arsenic toxicity induced endothelial dysfunction and dementia: pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors.

Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate & brain GSH levels along with increase in serum & brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia.

Chronic endothelin-1 treatment leads to heterologous desensitization of insulin signaling in 3T3-L1 adipocytes.

We recently reported that insulin and endothelin-1 (ET-1) can stimulate GLUT4 translocation via the heterotrimeric G protein G alpha q/11 and through PI3-kinase--mediated pathways in 3T3-L1 adipocytes. Because both hormones stimulate glucose transport through a common downstream pathway, we determined whether chronic ET-1 pretreatment would desensitize these cells to acute insulin signaling. We found that ET-1 pretreatment substantially inhibited insulin-stimulated 2-deoxyglucose uptake and GLUT4 translocation. Cotreatment with the ETA receptor antagonist BQ 610 prevented these effects, whereas inhibitors of G alpha i or G beta gamma were without effect. Chronic ET-1 treatment inhibited insulin-stimulated tyrosine phosphorylation of G alpha q/11 and IRS-1, as well as their association with PI3-kinase and blocked the activation of PI3-kinase activity and phosphorylation of AKT: In addition, chronic ET-1 treatment caused IRS-1 degradation, which could be blocked by inhibitors of PI3-kinase or p70 S6-kinase. Similarly, expression of a constitutively active G alpha q mutant, but not the wild-type G alpha q, led to IRS-1 degradation and inhibited insulin-stimulated phosphorylation of IRS-1, suggesting that the ET-1-induced decrease in IRS-1 depends on G alpha q/11 and PI3-kinase. Insulin-stimulated tyrosine phosphorylation of SHC was also reduced in ET-1 treated cells, resulting in inhibition of the MAPK pathway. In conclusion, chronic ET-1 treatment of 3T3-L1 adipocytes leads to heterologous desensitization of metabolic and mitogenic actions of insulin, most likely through the decreased tyrosine phosphorylation of the insulin receptor substrates IRS-1, SHC, and G alpha q/11.

Adenovirus-mediated overexpression of IRS-1 interacting domains abolishes insulin-stimulated mitogenesis without affecting glucose transport in 3T3-L1 adipocytes.

Activated insulin receptor (IR) interacts with its substrates, IRS-1, IRS-2, and Shc via the NPXY motif centered at Y960. This interaction is important for IRS-1 phosphorylation. Studies using the yeast two-hybrid system and sequence identity analysis between IRS-1 and IRS-2 have identified two putative elements, the PTB and SAIN domains, between amino acids 108 and 516 of IRS-1 that are sufficient for receptor interaction. However, their precise function in mediating insulin's bioeffects is not understood. We expressed the PTB and SAIN domains of IRS-1 in HIRcB fibroblasts and 3T3-L1 adipocytes utilizing replication-defective adenoviral infection to investigate their role in insulin signalling. In both cell types, overexpression of either the PTB or the SAIN protein caused a significant decrease in insulin-induced tyrosine phosphorylation of IRS-1 and Shc proteins, IRS-1-associated phosphatidylinositol 3-kinase (PI 3-K) enzymatic activity, p70s6k activation, and p44 and p42 mitogen-activated protein kinase (MAPK) phosphorylation. However, epidermal growth factor-induced Shc and MAPK phosphorylation was unaffected by the overexpressed proteins. These findings were associated with a complete inhibition of insulin-stimulated cell cycle progression. In 3T3-L1 adipocytes, PTB or SAIN expression extinguished IRS-1 phosphorylation with a corresponding 90% decrease in IRS-1-associated PI 3-K activity. p70s6k is a downstream target of PI 3-K, and insulin-stimulated p70s6k was inhibited by PTB or SAIN expression. Interestingly, overexpression of either PTB or SAIN protein did not affect insulin-induced AKT activation or insulin-stimulated 2-deoxyglucose transport, even though both of these bioeffects are inhibited by wortmannin. Thus, interference with the IRS-1-IR interaction inhibits insulin-stimulated IRS-1 and Shc phosphorylation, PI 3-K enzymatic activity, p70s6k activation, MAPK phosphorylation and cell cycle progression. In 3T3-L1 adipocytes, interference with the IR-IRS-1 interaction did not cause inhibition of insulin-stimulated AKT activation or glucose transport. These results indicate a bifurcation or subcompartmentalization of the insulin signalling pathway whereby some targets of PI 3-K (i.e., p70s6k) are dependent on IRS-1-associated PI 3-K and other targets (i.e., AKT and glucose transport) are not. IR-IRS-1 interaction is not essential for insulin's effect on glucose transport, and alternate, or redundant, pathways exist in these cells.

Molecular cloning of rat Wilms' tumor complementary DNA and a study of messenger RNA expression in the urogenital system and the brain.

In an effort to study the molecular basis of kidney development and carcinogenesis, we isolated complementary DNA clones of the rat homologue of the human Wilms' tumor gene, WT-1. When compared to the predicted sequence of the human WT-1 polypeptide, the rat WT-1 amino acid sequence is highly conserved (> 97%), except for the loss of one amino acid. In situ mRNA hybridization experiments localized WT-1 expression to the glomerular cells in the kidney during embryogenesis and the Sertoli cells of the testis. Similar to expression in humans and mice, WT-1 mRNA expression in the rat kidney and testis is developmentally regulated. In addition, two novel sites of specific high level WT-1 mRNA expression were seen. One is an area in the spinal cord where high level expression occurs throughout embryonic development. The second is the area postrema in the brain where localized expression continues through adulthood, suggesting a functional role for WT-1 in rat brain.

Wilms' tumor suppressor gene (WT1) is expressed in primary breast tumors despite tumor-specific promoter methylation.

We analyzed Wilms' tumor suppressor 1 (WT1) expression and its regulation by promoter methylation in a panel of normal breast epithelial samples and primary carcinomas. Contrary to previous reports, WT1 protein was strongly expressed in primary carcinomas (27 of 31 tumors) but not in normal breast epithelium (1 of 20 samples). Additionally, the WT1 promoter was methylated in 6 of 19 (32%) primary tumors, which nevertheless expressed WT1. The promoter is not methylated in normal epithelium. Thus, although tumor-specific methylation of WT1 is established in primary breast cancer at a low frequency, other transcriptional regulatory mechanisms appear to supercede its effects in these tumors. Our results demonstrate expression of WT1 in mammary neoplasia, and that WT1 may not have a tumor suppressor role in breast cancer.

A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1.

Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor and acts as a docking protein for Src homology 2 domain containing signaling molecules that mediate many of the pleiotropic actions of insulin. Insulin stimulation elicits serine/threonine phosphorylation of IRS-1, which produces a mobility shift on SDS-PAGE, followed by degradation of IRS-1 after prolonged stimulation. We investigated the molecular mechanisms and the functional consequences of these phenomena in 3T3-L1 adipocytes. PI 3-kinase inhibitors or rapamycin, but not the MEK inhibitor, blocked both the insulin-induced electrophoretic mobility shift and degradation of IRS-1. Adenovirus-mediated expression of a membrane-targeted form of the p110 subunit of phosphatidylinositol (PI) 3-kinase (p110CAAX) induced a mobility shift and degradation of IRS-1, both of which were inhibited by rapamycin. Lactacystin, a specific proteasome inhibitor, inhibited insulin-induced degradation of IRS-1 without any effect on its electrophoretic mobility. Inhibition of the mobility shift did not significantly affect tyrosine phosphorylation of IRS-1 or downstream insulin signaling. In contrast, blockade of IRS-1 degradation resulted in sustained activation of Akt, p70 S6 kinase, and mitogen-activated protein (MAP) kinase during prolonged insulin treatment. These results indicate that insulin-induced serine/threonine phosphorylation and degradation of IRS-1 are mediated by a rapamycin-sensitive pathway, which is downstream of PI 3-kinase and independent of ras/MAP kinase. The pathway leads to degradation of IRS-1 by the proteasome, which plays a major role in down-regulation of certain insulin actions during prolonged stimulation.

The acute and chronic stimulatory effects of endothelin-1 on glucose transport are mediated by distinct pathways in 3T3-L1 adipocytes.

We have recently shown that pretreatment with endothelin-1 (ET-1) for 20 min stimulates GLUT4 translocation in a PI3-kinase-dependent manner in 3T3-L1 adipocytes (Imamura, T. et al., J Biol Chem 274:33691-33695). This study presents another pathway by which ET-1 potentiates glucose transport in 3T3-L1 adipocytes. ET-1 treatment (10 nM) leads to approximately 2.5-fold stimulation of 2-deoxyglucose (2-DOG) uptake within 20 min, reaching a maximal effect of approximately 4-fold at approximately 6 h, and recovering almost to basal levels after 24 h. Insulin treatment (3 ng/ml) results in an approximately 5-fold increase in 2-DOG uptake at 1 h, and recovering to basal levels after 24 h. The ETA receptor antagonist, BQ 610, inhibited ET-1 induced glucose uptake both at 20 min and 6 h, whereas the ETB receptor antagonist, BQ 788, was without effect. Interestingly, ET-1 stimulated 2-DOG uptake at 6 h, not at 20 min, was almost completely blocked by the protein-synthesis inhibitor, cycloheximide and the RNA-synthesis inhibitor, actinomycin D, suggesting that the short-term (20 min) and long-term (6 h) effects of ET-1 involve distinct mechanisms. GLUT4 translocation assay showed that 20 min, but not 6 h, exposure to ET-1 led to GLUT4 translocation to the plasma membrane. In contrast, 6 h, but not 20 min, exposure to ET-1 increased expression of the GLUT1 protein, without affecting expression of GLUT4 protein. ET-1 induced 2-DOG uptake and GLUT1 expression at 6 h were completely inhibited by the MEK inhibitor, PD 98059, and partially inhibited by the PI3-kinase inhibitor, LY 294002, and the G alpha i inhibitor, pertussis toxin. The PLC inhibitor, U 73122, was without effect. These findings suggest that ET-1 induced GLUT1 protein expression is primarily mediated via MAPK, and partially via PI3K in 3T3-L1 adipocytes.

Persistent activation of phosphatidylinositol 3-kinase causes insulin resistance due to accelerated insulin-induced insulin receptor substrate-1 degradation in 3T3-L1 adipocytes.

Recently, we have reported that the overexpression of a membrane-targeted phosphatidylinositol (PI) 3-kinase (p110CAAX) stimulated p70S6 kinase, Akt, glucose transport, and Ras activation in the absence of insulin but inhibited insulin-stimulated glycogen synthase activation and MAP kinase phosphorylation in 3T3-L1 adipocytes. To investigate the mechanism of p110CAAX-induced cellular insulin resistance, we have now studied the effect of p110CAAX on insulin receptor substrate (IRS)-1 protein. Overexpression of p110CAAX alone decreased IRS-1 protein levels to 63+/-10% of control values. Insulin treatment led to an IRS-1 gel mobility shift (most likely caused by serine/threonine phosphorylation), with subsequent IRS-1 degradation. Moreover, insulin-induced IRS-1 degradation was enhanced by expression of p110CAAX (61+/-16% vs. 13+/-15% at 20 min, and 80+/-8% vs. 41+/-12% at 60 min, after insulin stimulation with or without p110CAAX expression, respectively). In accordance with the decreased IRS-1 protein, the insulin-stimulated association between IRS-1 and the p85 subunit of PI 3-kinase was also decreased in the p110CAAX-expressing cells, and IRS-1-associated PI 3-kinase activity was decreased despite the fact that total PI 3-kinase activity was increased. Five hours of wortmannin pretreatment inhibited both serine/threonine phosphorylation and degradation of IRS-1 protein. These results indicate that insulin treatment leads to serine/threonine phosphorylation of IRS-1, with subsequent IRS-1 degradation, through a PI 3-kinase-sensitive mechanism. Consistent with this, activated PI 3-kinase phosphorylates IRS-1 on serine/threonine residues, leading to IRS- 1 degradation. The similar finding was observed in IRS-2 as well as IRS-1. These results may also explain the cellular insulin-resistant state induced by chronic p110CAAX expression.

Cloning and expression of a human muscle phosphofructokinase cDNA.

The nucleotide sequence of a 2.86-kb cDNA clone containing the complete human muscle phosphofructokinase (PFK) protein-coding region was determined. It comprises 76 bp of 5'-untranslated sequence, 2340 bp encoding human muscle PFK polypeptide, and 399 bp of 3'-untranslated sequence plus a poly(A) tract. A retroviral vector was utilized to express the product of this coding sequence in mouse fibroblasts. The PFK-coding cDNA was shown to code for an enzymatically active polypeptide by immunoprecipitation analysis and DEAE-Sephadex A-25 chromatography.

Cloning and characterization of the human muscle phosphofructokinase gene.

A 35-kbp region of genomic DNA encoding the human muscle phosphofructokinase (HPFK-M) gene including all of the coding exons (1-22) plus 2.2-kbp of 5'-flanking sequence has been cloned. The exon boundaries are the same as has been observed for the rabbit muscle phosphofructokinase (RPFK-M), the human liver phosphofructokinase (HPFK-L), and the mouse liver phosphofructokinase (MPFK-L) genes. Characterization of the structure of the HPFK-M gene and its transcript in Epstein-Barr virus transformed B-cell lines derived from patients with glycogen storage disease type VII (GSDVII or Tarui's disease) demonstrated that this single-copy gene encodes a normal sized 3.0-kb transcript in the four cases examined. This suggests the lesion in these cases represents either a point mutation or possibly a small insertion or deletion resulting in the synthesis of a defective HPFK-M protein. Analysis of the 5'-flanking region demonstrated the presence of a functional promoter located within 114 nucleotides of a proposed transcription initiation site. This promoter was active in the human cervical carcinoma cell line, HeLa S3, the dedifferentiated human hepatoma cell line, HepG2.1, and the mouse myoblast cell line, C2C12, suggesting this promoter has a broad cell-type specificity. In addition, from the known HPFK-M cDNA sequences, this observation indicates that the HPFK-M gene has a second promoter located upstream from the genomic region isolated in this study.

N-ethylmaleimide blocks the modulatory effects of divalent cations and guanine nucleotides on the brain substance P receptor.

The binding of [3H]physalaemin ([3H]PHY) to rat brain substance P receptors is modulated by cations and guanine nucleotides. [3H]PHY binding in the presence of either monovalent or divalent cations (125 mM Na2SO4 or 2.5 mM MnCl2) shows a KD of 5.9 and 5.5 nM and a Bmax of 44.4 and 63.9 fmol/mg protein respectively. In the presence of both, there is a 2-fold increase in the affinity (KD 2.8 nM) and a 25-80% increase in the Bmax (81.6 fmol/mg protein). Addition of 100 microM GTP or Gpp(NH)p in either 125 mM Na2SO4 or 2.5 mM MnCl2 or both decreases the Bmax by 25-55%. However, the receptor affinity for [3H]PHY is not significantly altered by guanine nucleotides. N-Ethylmaleimide (NEM) irreversibly inhibits the receptor binding with an IC50 of 1.0 mM, demonstrating that SH groups play a critical role in the interaction of the ligand with the receptor. If the SP receptors are protected with 1 microM PHY, NEM irreversibly inhibits the effect of divalent cations and guanine nucleotides. Analysis of [3H]PHY binding in 125 mM Na2SO4, 2.5 mM MnCl2 on membranes that were protected with 1 microM PHY and then preincubated with NEM demonstrates a variable decline in receptor number and a 2-fold decrease in the affinity (KD, from 2.8 to 6.9 nM). These observations indicate the existence of a second class of SH groups that are essential for the interaction of divalent cations and guanine nucleotides with the receptor. The blockade of the modulatory effects of divalent cations and guanine nucleotides by NEM treatment further suggests that brain SP receptors are coupled to a guanine nucleotide binding regulatory protein.

Evidence of mucin secretion in human lung adenocarcinoma cell lines NCIH650 and NCIH2077 and effect of select secretagogues on mucin secretion.

Mucins comprise an important class of tumor-associated antigens. The objectives of the present study were (a) to establish an in vitro model system using human non-small cell lung adenocarcinoma cell lines NCIH650 and NCIH2077 (b) provide evidence that these cell lines secrete mucin in culture conditions and (c) investigate the effects of select secretagogues on mucin secretion. The cell lines were established in ACL-4 medium containing several growth factors and retinoic acid and 5% fetal calf serum. The high molecular weight glycoconjugates secreted in the culture medium were purified by ammonium sulfate precipitation and Superose 6 and Superose 12 FPLC chromatography. The purified high molecular weight glycoconjugate fraction and the carcinoma cells were shown to have mucin by dot blot, Western blot and immunohistochemical analysis, respectively, using specific antibodies to purified major mucin, HTM-1. Also, incorporation experiments with mucin precursor 3H-glucosamine demonstrated that the cells indeed synthesize high molecular weight mucins. The effects of secretagogues such as, 8-bromocyclic AMP, ionomycin, phorbol-12-myristate-13-acetate and neutrophil elastase on mucin secretion were also investigated. Only 8-bromocyclic AMP and neutrophil elastase influenced mucin secretion. These studies provided strong evidence that the lung adenocarcinoma cell lines secrete high molecular weight mucins in culture conditions and only two of the four tested secretagogues significantly increased mucin secretion. Thus, this in vitro model system may be useful in determining alterations in mucin structure, if any, in lung adenocarcinomas as well as in studying the regulation of mucin gene expression.

The osmotic shock-induced glucose transport pathway in 3T3-L1 adipocytes is mediated by gab-1 and requires Gab-1-associated phosphatidylinositol 3-kinase activity for full activation.

Osmotic shock treatment of 3T3-L1 adipocytes causes an increase in glucose transport activity and translocation of GLUT4 protein similar to that elicited by insulin treatment. Insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, however, activation by osmotic shock was only partially blocked. Additionally, we have found that the newly identified insulin receptor substrate Gab-1 (Grb2-associated binder-1) is tyrosine-phosphorylated following sorbitol stimulation. Treatment of cells with the tyrosine kinase inhibitor genistein inhibited osmotic shock-stimulated Gab-1 phosphorylation as well as shock-induced glucose transport. Furthermore, pretreatment with the selective Src family kinase inhibitor PP2 completely inhibited the ability of sorbitol treatment to cause tyrosine phosphorylation of Gab-1. We have also shown that microinjection of anti-Gab-1 antibody inhibits osmotic shock-induced GLUT4 translocation. Furthermore, phosphorylated Gab-1 binds and activates phosphatidylinositol 3-kinase (PI3K) in response to osmotic shock. The PI3K activity associated with Gab-1 was 82% of that associated with anti-phosphotyrosine antibodies, indicating that Gab-1 is the major site for PI3K recruitment following osmotic shock stimulation. Although wortmannin only causes a partial block of osmotic shock-stimulated glucose uptake, wortmannin completely abolishes Gab-1 associated PI3K activity. This suggests that other tyrosine kinase-dependent pathways, in addition to the Gab-1-PI3K pathway, contribute to osmotic shock-mediated glucose transport. To date, Gab-1 is the first protein identified as a member of the osmotic shock signal transduction pathway.

A rodent model for Wilms tumors: embryonal kidney neoplasms induced by N-nitroso-N'-methylurea.

Embryonal kidney cell tumors develop in rats given the alkylating agent N-nitroso-N'-methylurea as neonates. These tumors resemble the childhood Wilms tumors in their histopathology. Deletions and mutations in the Wilms tumor suppressor gene, WT1, are present in up to 6% of childhood nephroblastomas. To investigate the role of WT1 in rat kidney tumorigenesis, we studied the genetic alterations in WT1 and its target genes. Point mutations were found in WT1 cDNA in 7 of 18 kidney tumors. Mesenchymal tumors contained G-->A transition mutations in codons 128, 364, and 372, typical of the methylating action of N-nitroso-N'-methylurea on DNA. Each of the four nephroblastomas contained the same T-->A mutation at codon 111 of WT1, reflective of transversion mutagenesis by N-nitroso-N'-methylurea in vivo. Like Wilms tumors, mRNA levels of WT1, IGF2, Pax-2, and MK genes were higher than newborn kidney in the majority of the tumors. The histopathology of the rat kidney tumors and the genetic alterations are reminiscent of those observed in Wilms tumors, establishing this as a relevant model system for the human disease.

RNA editing in the Wilms' tumor susceptibility gene, WT1.

Rat kidney WT1 cDNAs contain either a thymidine or a cytosine residue at position 839. Genomic WT1 DNA contains only T839. To explain these results, we propose the WT1 transcript undergoes RNA editing in which U839 is converted to C, resulting in the replacement of leucine 280 in WT1 by proline. RNA editing at the same nucleotide was observed in WT1 cDNAs from human testis. In functional assays, the WT1-leucine280 polypeptide repressed the EGR-1 promoter in in vitro assays approximately 30% more efficiently than WT1-proline. Edited WT1-C839 mRNA was barely detectable in neonatal kidney, whereas adult rat kidneys contained both U839 and C839-WT1 mRNA, suggesting a role for the two protein isoforms in growth and differentiation.

Comparison of alterations in insulin signalling pathway in adipocytes from Type II diabetic pregnant women and women with gestational diabetes mellitus.

AIMS/HYPOTHESIS: The cellular mechanisms for the insulin resistance in pregnancy and gestational diabetes mellitus are not known. The membrane protein plasma cell glycoprotein PC-1 has been identified as an inhibitor of insulin receptor tyrosine kinase activity and could have a role in insulin resistance. This study aimed to examine the effects of insulin on glucose transport and changes in insulin receptor tyrosine phosphorylation, IRS-1 and PC-1. METHODS: Adipocytes were obtained either during elective cesarean section from three groups of subjects (Type II diabetic pregnant women ( n=6) women with gestational diabetes mellitus ( n=10) and pregnant women with normal glucose tolerance ( n=6) as pregnant control subjects) or during elective gynaecological surgery from non-pregnant ( n=6) control subjects. RESULTS: Insulin stimulated glucose transport was reduced by 50% in women with gestational diabetes mellitus and 70% in pregnant women with Type II diabetes, compared to the non-pregnant control subjects. After maximal insulin stimulation of adipocytes, IRTK phosphorylation was reduced by 29.5% in women with gestational diabetes mellitus and 44.5% in women with Type II diabetes, compared to the non-pregnant control subjects. We also found that IRS-1 phosphorylation was reduced by 32% and 48%, respectively. On the other hand, PC-1 content in adipocytes in women with gestational diabetes mellitus increased by 320% and 668% in Type II diabetic women, compared to the non-pregnant control subjects. CONCLUSIONS/INTERPRETATION: Our results indicate that women with gestational diabetes mellitus and Type II diabetes have increased PC-1 content and suggest that this could contribute to lower phosphorylation levels of IRTK and IRS-1. Furthermore, these postreceptor defects in insulin signalling pathway are greater in both groups compared to the women with normal pregnancy. However, results from women with Type II diabetes show that pre-existing insulin resistance lead to an even greater deterioration of the signalling pathway.

The tumor suppressor PTEN negatively regulates insulin signaling in 3T3-L1 adipocytes.

PTEN is a tumor suppressor with sequence homology to protein-tyrosine phosphatases and the cytoskeleton protein tensin. PTEN is capable of dephosphorylating phosphatidylinositol 3,4, 5-trisphosphate in vitro and down-regulating its levels in insulin-stimulated 293 cells. To study the role of PTEN in insulin signaling, we overexpressed PTEN in 3T3-L1 adipocytes approximately 30-fold above uninfected or control virus (green fluorescent protein)-infected cells, using an adenovirus gene transfer system. PTEN overexpression inhibited insulin-induced 2-deoxy-glucose uptake by 36%, GLUT4 translocation by 35%, and membrane ruffling by 50%, all of which are phosphatidylinositol 3-kinase-dependent processes, compared with uninfected cells or cells infected with control virus. Microinjection of an anti-PTEN antibody increased basal and insulin stimulated GLUT4 translocation, suggesting that inhibition of endogenous PTEN function led to an increase in intracellular phosphatidylinositol 3,4,5-trisphosphate levels, which stimulates GLUT4 translocation. Further, insulin-induced phosphorylation of downstream targets Akt and p70S6 kinase were also inhibited significantly by overexpression of PTEN, whereas tyrosine phosphorylation of the insulin receptor and IRS-1 or the phosphorylation of mitogen-activated protein kinase were not affected, suggesting that the Ras/mitogen-activated protein kinase pathway remains fully functional. Thus, we conclude that PTEN may regulate phosphatidylinositol 3-kinase-dependent insulin signaling pathways in 3T3-L1 adipocytes.

Alternative splicing of the transcript encoding the human muscle isoenzyme of phosphofructokinase.

The genomic DNA encoding human muscle phosphofructokinase (HPFK-M) exons VII to X has been cloned and the coding regions have been sequenced. The intron/exon boundaries are located at the same positions as those identified for the rabbit phosphofructokinase-M gene (Lee, C. -P., Kao, M. -C., French, B. A., Putney, S. D., and Chang, S. H. (1987) J. Biol. Chem. 262, 4195-4199). A HPFK-M cDNA clone lacking the sequences corresponding to exon IX was isolated from a human fibroblast (IMR-90) library, suggesting that the HPFK-M transcript may be alternatively spliced. Exon IX is 93 nucleotides in length, and the absence of this sequence from the HPFK-M transcript would generate an RNA coding for a HPFK-M-related polypeptide lacking 31 amino acids compared with the HPFK-M polypeptide. HPFK-M transcripts approximately 3.0 kilobases in length are expressed in a tissue-specific manner with high levels in cell lines and skeltal muscle tissue and very low levels in peripheral blood mononuclear cells and liver tissue. Characterization of the structure of these HPFK-M transcripts by nuclease S1 and polymerase chain reaction analysis demonstrated that all the cell lines and tissues examined expressed the alternatively spliced transcript in addition to the transcript coding for the enzymatically functional HPFK-M polypeptide.

Inhibition of phosphatidylinositol 3-kinase activity by adenovirus-mediated gene transfer and its effect on insulin action.

Phosphatidylinositol 3-kinase (PI 3-K) is implicated in cellular events including glucose transport, glycogen synthesis, and protein synthesis. It is activated in insulin-stimulated cells by binding of the Src homology 2 (SH2) domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1), and, others. We have previously shown that IRS-1-associated PI 3-kinase activity is not essential for insulin-stimulated glucose transport in 3T3-L1 adipocytes, and that alternate pathways exist in these cells. We now show that adenovirus-mediated overexpression of the p85N-SH2 domain in these cells behaves in a dominant-negative manner, interfering with complex formation between endogenous PI 3-K and its SH2 binding targets. This not only inhibited insulin-stimulated IRS-1-associated PI 3-kinase activity, but also completely blocked anti-phosphotyrosine-associated PI 3-kinase activity, which would include the non-IRS-1-associated activity. This resulted in inhibition of insulin-stimulated glucose transport, glycogen synthase activity and DNA synthesis. Further, Ser/Thr phosphorylation of downstream molecules Akt and p70 S6 kinase was inhibited. However, co-expression of a membrane-targeted p110(C) with the p85N-SH2 protein rescued glucose transport, supporting our argument that the p85N-SH2 protein specifically blocks insulin-mediated PI 3-kinase activity, and, that the signaling pathways downstream of PI 3-kinase are intact. Unexpectedly, GTP-bound Ras was elevated in the basal state. Since p85 is known to interact with GTPase-activating protein in 3T3-L1 adipocytes, the overexpressed p85N-SH2 peptide could titrate out cellular GTPase-activating protein by direct association, such that it is unavailable to hydrolyze GTP-bound Ras. However, insulin-induced mitogen-activated protein kinase phosphorylation was inhibited. Thus, PI 3-kinase may be required for this action at a step independent of and downstream of Ras. We conclude that, in 3T3-L1 adipocytes, non-IRS-1-associated PI 3-kinase activity is crucial for insulin's metabolic signaling, and that overexpressed p85N-SH2 protein inhibits a variety of insulin's ultimate biological effects.

Membrane-targeted phosphatidylinositol 3-kinase mimics insulin actions and induces a state of cellular insulin resistance.

Phosphatidylinositol (PI) 3-kinase plays an important role in various insulin-stimulated biological responses including glucose transport, glycogen synthesis, and protein synthesis. However, the molecular link between PI 3-kinase and these biological responses is still unclear. We have investigated whether targeting of the catalytic p110 subunit of PI 3-kinase to cellular membranes is sufficient and necessary to induce PI 3-kinase dependent signaling responses, characteristic of insulin action. We overexpressed Myc-tagged, membrane-targeted p110 (p110(CAAX)), and wild-type p110 (p110(WT)) in 3T3-L1 adipocytes by adenovirus-mediated gene transfer. Overexpressed p110(CAAX) exhibited approximately 2-fold increase in basal kinase activity in p110 immunoprecipitates, that further increased to approximately 4-fold with insulin. Even at this submaximal PI 3-kinase activity, p110(CAAX) fully stimulated p70 S6 kinase, Akt, 2-deoxyglucose uptake, and Ras, whereas, p110(WT) had little or no effect on these downstream effects. Interestingly p110(CAAX) did not activate MAP kinase, despite its stimulation of p21(ras). Surprisingly, p110(CAAX) did not increase basal glycogen synthase activity, and inhibited insulin stimulated activity, indicative of cellular resistance to this action of insulin. p110(CAAX) also inhibited insulin stimulated, but not platelet-derived growth factor-stimulated mitogen-activated protein kinase phosphorylation, demonstrating that the p110(CAAX) induced inhibition of mitogen-activated protein kinase and insulin signaling is specific, and not due to some toxic or nonspecific effect on the cells. Moreover, p110(CAAX) stimulated IRS-1 Ser/Thr phosphorylation, and inhibited IRS-1 associated PI 3-kinase activity, without affecting insulin receptor tyrosine phosphorylation, suggesting that it may play an important role as a negative regulator for insulin signaling. In conclusion, our studies show that membrane-targeted PI 3-kinase can mimic a number of biologic effects normally induced by insulin. In addition, the persistent activation of PI 3-kinase induced by p110(CAAX) expression leads to desensitization of specific signaling pathways. Interestingly, the state of cellular insulin resistance is not global, in that some of insulin's actions are inhibited, whereas others are intact.