Inositol Phosphate Glycan Research Articles

An anionic inositol phosphate glycan pseudotetrasaccharide exhibits high insulin-mimetic activity in rat adipocytes

Inositol phosphate glycan pseudotetrasaccharides consisting of man-(α1-6)-man-(α1-4)-glcN-(α,β1-6)- myo-inositol-1,2-cyclic phosphate possessing a sulfate group at either O-6 (compounds 3α,β) or O-2 (compounds 4α,β) of the terminal mannose have been prepared. Compound 4α was able to stimulate lipogenesis in native rat adipocytes to 78% of the maximal insulin response (MIR) with an EC 50 of 1.1 μM. The other compounds exhibited lower maximal stimulations (47–63% MIR) and higher EC 50 values (9.5–10.6 μM).

A fluorescent inositol phosphate glycan stimulates lipogenesis in rat adipocytes by extracellular activation alone

The chemical synthesis of 2,6-dideoxy-2-amino-6-mercaptoglucopyranosyl-(α1–6)- myo-inositol 1,2-cyclic phosphate and its conjugation with a lucifer yellow derivative are reported. The resulting fluorescent IPG analogue was able to stimulate lipogenesis in rat adipocytes despite the fact that it was not internalized into the cell. The results demonstrate that internalization of the IPG is not required for manifestation of its insulin-like effects.

Glycosylphosphatidylinositol (GPI) hydrolysis by transforming growth factor-beta1 (TGF-beta1) as a potential early step in the inhibition of epithelial cell proliferation.

Glycosylphosphatidylinositol (GPI) was previously identified in rabbit articular chondrocytes as being a precursor of inositolphosphate glycan (IPG), released upon (Transforming Growth Factor-beta) (TGF-beta) exposure, and capable of mimicking the proliferative effects of the growth factor. Here, using mink lung epithelial cells (CCL 64), which are known to be growth-inhibited by TGF-beta, we studied the potential role of GPI-derived molecules in the antiproliferative effect of TGF-beta1. We first identified an endogenous pool of GPI material and three different anionic forms of IPG in epithelial cells pre-labeled with [3H]glucosamine. Shortly (8 min) after TGF-beta1 addition, the cells responded by a rapid and transient hydrolysis of GPI, accompanied by the release of the most anionic form of IPG. This TGF-beta-released IPG, after partial purification, was shown to decrease the proliferation of CCL 64 cells. Moreover, anti-IPG antibodies reduced the effects of TGF-beta and blocked the effects of partially purified IPG. These data strongly suggest that GPI hydrolysis may be an early step of the TGF-beta signalling pathway involved in growth inhibition of epithelial cells.

Purification and analysis of the neutral glycan moiety of glycosyl phosphatidylinositol from porcine thyroid cells.

Metabolic labelling of inositolphosphate glycan with radioactive precursors is not sufficient to characterize and assess the involvement of the glycosyl phosphatidylinositol/inositolphosphate glycan (GPI/IPG) system in porcine thyroid cell signal transduction machinery. A protocol is described for the isolation and purification of free GPI using differential polarity of lipids and sequential thin layer chromatography. The purification until homogeneity of GPI constitutes a required step for gas chromatographic analysis. Next, successive chemical treatments allowed us to remove the neutral glycan moiety of thyroidal GPI, and its composition was obtained by gas chromatography. The proposed structure is consistent with data available for GPI anchor, but differs from compositional analysis data reported for insulin-sensitive GPI. Our results support the existence in porcine thyroid cells of the GPI/IPG system, which can take part in TSH-dependent signal transduction processes.

Glycosyl phosphatidylinositol (GPI)/inositolphosphate glycan (GPI): An intracellular signalling system involved in the control of thyroid cell proliferation

In porcine thyrocytes, TSH alone does not induce cell growth. Recently, it has been demonstrated that acute stimulation by TSH of porcine thyrocytes leads to release an inositolphosphate glycan (IPG) described as a putative second messenger for various growth factors in different cell types. IPG isolated from porcine thyrocytes induces proliferation of fibroblasts EGFR T17 and porcine thyrocytes. In porcine thyrocytes we have confirmed that cell growth requires the presence of both TSH and insulin. This effect is reproduced by 8-bromo cyclic AMP suggesting a mediation by intracellular cyclic AMP. Cooperative effects between 8-bromo cyclic AMP and IPG have also been evidenced and are in favour of a crosstalk between distinct signalling pathways.

An inositolphosphate glycan released by TGF-β mimics the proliferative but not the transcriptional effects of the factor and requires functional receptors

Transforming growth factor-β 1 (TGF-β 1) is a multifunctional polypeptide that regulates a number of cellular processes including cell growth and deposition of extracellular matrix protein. Despite the fact that the signal transduction by TGF-β has been intensively studied, the molecular mechanisms of that pathway are not clear. We have studied the possibility that an inositolphosphate glycan (IPG) is involved in transmission of the TGF-β 1 signal. We show that TGF-β 1 induces IPG release in both rabbit articular chondrocytes (RAC), which are growth stimulated by the factor and Mv 1Lu cell line, which is growth inhibited. This release requires functional TGF-β heteromeric receptors in these two cell types. We also demonstrate that IPG mimics TGF-β 1-induced growth stimulation in mesenchymal cells (+100%) and growth inhibition in epithelial cells (−80%). Moreover TGF-β receptor I (TβR-I) is not required for inhibition of proliferation induced by IPG since derivated mutants of the Mv 1Lu cell line lacking TβR-I intracellular domain (R-1B) are significantly inhibited (−65%). Additionally, we show that IPG does not take part in the signalling pathway that leads to activation of matrix gene transcription. These results suggest that TGF-β effects on growth regulation and extracellular matrix synthesis implicate two different signalling pathways, IPG being only involved in growth regulation.

An inositol phosphate glycan derived from a Trypanosoma brucei glycosyl phosphatidylinositol promotes protein dephosphorylation in rat epididymal adipocytes

Some of the acute actions of insulin may be mediated by an enzyme-modulating inositol phosphate glycan, produced by the insulin-sensitive hydrolysis of a glycosyl phosphatidylinositol that is structurally similar to a membrane protein anchor. An inositol glycan fragment from the structurally characterized Trypanosoma brucei variant surface glycoprotein glycosyl phosphatidylinositol anchor inhibits isoproterenol-stimulated lipolysis in intact rat epididymal adipocytes in a manner mechanistically similar to that of insulin. To explore these effects in more detail, we evaluated the effects of this glycan on protein phosphorylation. Isoproterenol stimulates the phosphorylation of a 70-kilodalton (kDa) protein in these cells. Like insulin, the glycan fragment specifically attenuates the phosphorylation state of the phosphoprotein. In purified adipocyte cytosol, the glycan stimulates the dephosphorylation on serine residues of a 70-kDa protein in a time- and dose-dependent fashion. The glycan-dependent dephosphorylation of the 70-kDa phosphoprotein is unaffected by addition of trifluoroperazine, an inhibitor of serine/threonine phosphatase-2B, but is blocked by the addition of okadaic acid, an inhibitor of serine/threonine phosphatase-1 and -2A. The differential sensitivities of this dephosphorylation reaction to polycations, which activate phosphatase-2A, and phosphorylated inhibitor 1, which blocks phosphatase-1, suggest that dephosphorylation of the 70-kDa protein results from the specific activation of a type 1 serine/threonine phosphatase in adipocytes, providing a mechanistic basis for the insulin-mimetic effects of the inositol glycan.

An inositol phosphate glycan derived from a Trypanosoma brucei glycosyl phosphatidylinositol promotes protein dephosphorylation in rat epididymal adipocytes.

Some of the acute actions of insulin may be mediated by an enzyme-modulating inositol phosphate glycan, produced by the insulin-sensitive hydrolysis of a glycosyl phosphatidylinositol that is structurally similar to a membrane protein anchor. An inositol glycan fragment from the structurally characterized Trypanosoma brucei variant surface glycoprotein glycosyl phosphatidylinositol anchor inhibits isoproterenol-stimulated lipolysis in intact rat epididymal adipocytes in a manner mechanistically similar to that of insulin. To explore these effects in more detail, we evaluated the effects of this glycan on protein phosphorylation. Isoproterenol stimulates the phosphorylation of a 70-kilodalton (kDa) protein in these cells. Like insulin, the glycan fragment specifically attenuates the phosphorylation state of the phosphoprotein. In purified adipocyte cytosol, the glycan stimulates the dephosphorylation on serine residues of a 70-kDa protein in a time- and dose-dependent fashion. The glycan-dependent dephosphorylation of the 70-kDa phosphoprotein is unaffected by addition of trifluoroperazine, an inhibitor of serine/threonine phosphatase-2B, but is blocked by the addition of okadaic acid, an inhibitor of serine/threonine phosphatase-1 and -2A. The differential sensitivities of this dephosphorylation reaction to polycations, which activate phosphatase-2A, and phosphorylated inhibitor 1, which blocks phosphatase-1, suggest that dephosphorylation of the 70-kDa protein results from the specific activation of a type 1 serine/threonine phosphatase in adipocytes, providing a mechanistic basis for the insulin-mimetic effects of the inositol glycan.

Erythropoietin stimulates glycosylphosphatidylinositol hydrolysis in rat erythroid progenitor cells and inositolphosphate glycan modulates their proliferation.

The involvement of a glycosylphosphatidylinositol/inositolphosphate glycan (GPI/IPG) system in the erythropoietin (Epo) signal transduction was investigated. Endogenous GPI was evidenced in extracts of normal Epo-responsive cells after incorporation of [3H]glucosamine, [3H]inositol and [32P]orthophosphate. Incubation of these cells with Epo produced a rapid and transient hydrolysis of GPI with parallel release of IPG. IPG production was Epo dose dependent and the maximal effect was obtained with the same concentration of Epo which gave the maximal mitogenic effect, i.e. 1 U/ml. The number and size of erythroid colonies (CFU-E) were increased by the addition of purified rat erythroid IPG to the culture medium, but not to the same extent as with a maximal Epo treatment. Exogenous IPG effect was dose dependent. In the presence of suboptimal Epo concentrations, IPG has been found to potentiate Epo-induced CFU-E growth. These results support the hypothesis that a GPI/IPG based signal transduction system may be involved in Epo-induced cell proliferation.

Insulin resistance in genetically obese (fa/fa) rats: changes in the glycosyl-phosphatidylinositol signaling system in isolated hepatocytes.

In different types of mammalian cells, insulin has been shown to promote the release of an inositol phosphate glycan (InsP-glycan) through the hydrolysis of a glycosyl-phosphatidylinositol (glycosyl-PtdIns). This InsP-glycan, which has been demonstrated to be taken up by intact cells, may mediate some of the biological effects of insulin. We have investigated how the insulin resistance expressed in genetically obese (fa/fa) rats affects the glycosyl-PtdIns signaling system in isolated hepatocytes compared to what occurs in hepatocytes isolated from lean (Fa/-) rats. The hepatocyte content of glycosyl-PtdIns was reduced by about 30% in obese rats, with respect to that measured in lean rats (2553 +/- 138 vs. 3334 +/- 115 dpm/mg protein; P < 0.01; n = 5). This reduction was accompanied by a marked blockade of the insulin-mediated glycosyl-PtdIns hydrolysis as well as a decrease (approximately 30%) in the rate of InsP-glycan uptake by the isolated liver cells. Obese Zucker rat hepatocytes also showed a significant decrease in the effects of both insulin and InsP-glycan on the stimulation of glycogen synthesis and the activation of glycogen synthase compared to hepatocytes isolated from lean rats. Our results demonstrate that genetic obesity in Zucker (fa/fa) rats is associated with an impairment of the glycosyl-PtdIns-dependent insulin signaling system.

Different phosphorylated forms of inositolphosphate glycan could be involved in the transforming growth factor-gb 1 (TGF-β1) signalling pathway

Labelling with [ 3H]glucosamine was used to prepare a transforming growth factor-β1 (TGF-β1)-sensitive glycosylphosphatidylinositol (GPI) from monolayer cultures of rabbit articular chondrocytes (RAC), which may be involved in control of the cell cycle. The polar headgroup of this glycosylphosphatidylinositol was generated by both phosphatidylinositol-specific phospholipase C (PI-PLC) and pronase E digestion. The molecule emerged in only one peak on a Dowex AG1-X8 chromatogram, eluted at 0.1 N ammonium formate. In contrast, similar experiments performed on cellular extract from cultures previously labelled with [ 3h]glucosamine displayed four radioactive peaks diluting at 0.1, 0.2, 0.5 and 1 N ammonium formate, respectively. Evidence that the eluting position of these peaks was dependent on the number of phosphate residues present in each fraction was demonstrated by both [ 32P]phosphorus labelling and change in the position of alkaline phosphatase-induced shift in elution volume. We also demonstrated that the GPI-derived inositolphosphate glycan (IPG) could be hyperphosphorylated into the cell under the action of a kinase whose activity was enhanced by TGF-β1 itself. We have also shown that all of these IPG forms could mimic the TGF-β-induced increase of DNA replication rate of RAC, with a higher activity for peaks III and IV than peaks I and II.

Multiple regulation of S14 gene expression during brown fat differentiation.

S14 is a gene known to be under thyroid hormone control. Its mRNA concentration is very high in lipogenic tissues, and although the precise function of the protein is still unknown, indirect data suggest its implication in triglyceride synthesis. S14 gene expression is up-regulated by thyroid hormone in liver, white adipose tissue, and lactating mammary gland. However, in brown fat, the level of this sequence is increased 3-fold in the hypothyroid animal. We have used primary cultures of brown preadipocytes differentiated to fully mature brown adipocytes to investigate the influence of cellular differentiation and hormonal stimulation on S14 gene expression. Steady state levels of S14 mRNA rose from nondetectable levels in preadipocytes to reach a maximum in fully mature adipocytes. Treatment of brown adipocytes cultures with T3 did induce S14 gene expression. This induction reflects in part a posttranscriptional stabilization of the messenger by T3. Insulin, insulin-like growth factor-I, and inositol phosphate-glycan also increase the level of S14 mRNA. Norepinephrine (NE) plays a major role in the regulation of S14 gene, and 24 h after its addition, NE elicited a 20-fold decrease in mRNA S14 concentrations. An elevated intracellular concentration of cAMP is a strong negative effector of S14 gene expression, and neither NE nor cAMP action is totally overcome by T3. As happens in vivo, glucose is a potent stimulator of S14 mRNA; however, there is a lag time of several hours before its effects can be detected. The increase in S14 gene expression with the maturation stage of the cell suggests an important role for S14 in adipocyte differentiation.

IPG (inositolphosphate glycan) as a cellular signal for TGF‐β1 modulation of chondrocyte cell cycle

The knowledge of transforming growth factor (TGF)-beta receptors has greatly progressed in the recent years. TGF-beta receptors type I and II have been implicated in the modulation of cell proliferation, whereas type III (betaglycan) may act as a component presenting TGF-beta to its signaling receptors. In addition, four other proteins that bind TGF-beta 1 or TGF-beta 2 have been recently identified in some cell lines, three being anchored to the membrane through a glycosylphosphatidylinositol (GPI). Despite this knowledge, the molecular mechanism of signal transduction through the TGF-beta receptors remain an enigma. TGF-beta family does not signal via any of the classical pathways. As GPI anchors of membrane proteins have been implicated in the transduction of some hormonal effects, we investigated the putative role of GPI in signaling the TGF-beta effects on the proliferation of rabbit articular chondrocytes (RAC). We previously showed that TGF-beta 1 increased DNA replication rate of RAC, with a recruitment of cells in G2/M followed by a subsequent mitosis wave. Here, we find that the factor causes specific GPI hydrolysis, with correlated increase of inositolphosphate glycan (IPG). This effect was specifically inhibited by antibodies that bind TGF-beta 1. Using [3H]-inositol labeling and Triton X-114 extraction, we demonstrate that a hydrophobic material from the membrane is cleaved by treatment of cell cultures with phosphatidylinositol specific phospholipase C (PI-PLC) or by exposure to TGF-beta, supporting that a PI-anchored molecule gives rise to IPG by TGF-beta-induced hydrolysis. The biological relevance of this hydrolysis was demonstrated by the enhancing effect of purified IPG on the DNA synthesis rate, which mimicked the TGF-beta action. These results demonstrate that IPG could be an early messenger in the cellular signaling that mediates the effect of TGF-beta on RAC growth.

Changes in the insulin-sensitive glycosyl-phosphatidyl-inositol signalling system with aging in rat hepatocytes.

An inositol-phosphate glycan (InsP glycan), which is the polar head group of an insulin-sensitive glycosyl-phosphatidylinositol (glycosyl-PtdIns), has been reported to mimic some insulin actions when added to different types of cells. In connection with this, a specific, time-dependent and energy-dependent transport system for this InsP glycan has been identified in isolated rat hepatocytes [Alvarez, J. F., Sánchez-Arias, J. A., Guadaño, A., Estevez, F., Varela, I., Felíu, J. E. & Mato, J.M. (1991) Biochem. J. 274, 369-374]. Here we have investigated the glycosyl-PtdIns-dependent insulin-signalling system in hepatocytes isolated from either 3-month-old or 24-month-old rats. Aging reduced the stimulatory effect of insulin on [U-14C]glucose incorporation into glycogen, caused a significant decrease in basal glycosyl-PtdIns levels and blocked the insulin-mediated hydrolysis of this lipid. In 24-month-old rats, we also observed a diminution in the rate of hepatocyte InsP-glycan uptake and a marked reduction of the stimulatory effect of this compound on glycogen synthesis. These results support the hypothesis that insulin resistance associated with aging is accompanied by an impairment of the glycosyl-PtdIns-dependent cellular signalling system.

IPG (inositolphosphate glycan) as a cellular signal for TGF β modulation of chondrocyte cell cycle

IPG (inositolphosphate glycan) as a cellular signal for TGF β modulation of chondrocyte cell cycle

Partial characterization of a glycosylphosphatidyl-inositol (GPI) in porcine thyroid cultured cells

The aim of this study was to provide information on the structure of glycosylphosphatidylinositol (GPI) and to characterize this novel phospholipid isolated from pig thyroid. We investigated the incorporation of different radioactive precursors: [ 3H]glucosamine, [ 3H]inositol, [ 3H]oleic acid, [ 32P]orthophosphate and demonstrated the presence of these moieties in the structure of GPI. After labelling and hydrochloric acid hydrolysis, the major part of radioactivity comigrated with glucosamine used as marker. Cleavage of GPI by nitrous acid deamination indicated the presence of a glycosidic bond between the lipid moiety and glucosamine. The fatty acid composition of diacylglycerol was also studied by gas chromatography. Oleic acid was found preferentially to other fatty acids. In a previous paper we reported that a chronic treatment with 0.1 mU/ml thyrotropin (TSH) of thyroid cultured cells for 3 days produced a large increase in the turnover rate of GPI and concomitantly the release of a water-soluble product of GPI hydrolysis: an inositolphosphateglycan (IPG). These findings confirm that GPI may play a role in membrane signalling systems and thyroid cell regulation.

Impairment of glycosyl-phosphatidylinositol-dependent insulin signaling system in isolated rat hepatocytes by streptozotocin-induced diabetes.

The addition to different types of cells of an inositol-phosphate glycan, generated by the phospholipase C-catalyzed hydrolysis of a insulin-sensitive glycosyl-phosphatidylinositol (glycosyl-PI), mimics some of the biological effects of this hormone. Recently, a specific, time-, dose-, and energy-dependent transport system for this inositol-phosphate glycan has been identified in isolated rat hepatocytes. Here, we show that streptozotocin-induced diabetes mellitus reduced (by about 60%) the basal content of the insulin-sensitive glycosyl-PI in isolated rat hepatocytes. Moreover, streptozotocin-induced diabetes blocked the hydrolysis of the glycosyl-PI in response to insulin, diminished inositol phosphate-glycan uptake by the hepatocytes, and abolished the stimulatory effect of this compound on glycogen synthesis. All these metabolic changes caused by streptozotocin administration were reversed by treatment of the animals with insulin. Our results support the hypothesis that insulin resistance in streptozotocin-induced diabetic rats is related to the impairment of glycosyl-PI metabolism.

An inositol phosphate glycan derived from a Trypanosoma brucei glycosyl-phosphatidylinositol mimics some of the metabolic actions of insulin.

Some of the acute actions of insulin may be mediated by an enzyme-modulating inositol phosphate glycan, produced by the insulin-sensitive hydrolysis of glycosyl-phosphatidylinositol (GPI) that is structurally similar to a membrane protein anchor. An inositol glycan fragment from the structurally characterized Trypanosoma brucei variant surface glycoprotein GPI anchor is evaluated for insulin-mimetic antilipolytic activity. The fragment specifically and dose-dependently inhibits isoproterenol-stimulated lipolysis. Like the effect of insulin, glycan-induced antilipolysis is blocked by the low Km cAMP phosphodiesterase inhibitor imazodan (CI-914) and the serine/threonine phosphatase inhibitor, okadaic acid, suggesting that the activation of both cAMP phosphodiesterase and serine/threonine protein phosphatases are necessary. Moreover, this fragment causes a specific and dose-dependent inhibition of both microsomal glucose-6-phosphatase (EC 3.1.3.9) and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activity. Additionally, direct addition of the glycan to hepatocytes caused marked inhibition of glucose production from pyruvate. These results suggest that the direct modification of the activities of these two gluconeogenic enzymes by an inositol glycan may play a role in the inhibition of glucose output by insulin and provide the first evidence for the insulin-mimetic properties of a chemically characterized inositol glycan.

Functional analysis of T-cell mutants defective in the biosynthesis of glycosylphosphatidylinositol anchor. Relative importance of glycosylphosphatidylinositol anchor versus N-linked glycosylation in T-cell activation.

The glycosylphosphatidylinositol (GPI) anchor, potentially capable of generating a number of second messengers, such as diacylglycerol, phosphatidic acid, and inositol phosphate glycan, has been postulated to be involved in signal transduction in various cell types, including T-cells. We have identified a panel of T-cell hybridoma mutants that are defective at various steps of GPI anchor biosynthesis. Since they were derived from a functional T-T hybridoma, we were able to determine the precise role of the GPI anchor in T-cell activation. Two mutants were chosen for this analysis. The first mutant is defective at the first step of GPI anchor biosynthesis, i.e. in the transfer of N-acetylglucosamine to a phosphatidylinositol acceptor. Thus, it cannot form any GPI precursors or GPI-like compounds. Interestingly, this mutant can be activated by antigen, superantigen, and concanavalin A in a manner comparable to the wild-type hybridoma. These data strongly suggest that the GPI anchor, its precursor, or its potential cleavage product, inositol phosphate glycan, is not required for the early phase of T-cell activation. The second mutant is able to synthesize the first two GPI precursors, but is not able to add mannose residues to them due to a deficiency in dolichol-phosphate-mannose (Dol-P-Man) biosynthesis. Unexpectedly, all of the Dol-P-Man mutants are defective in activation by antigen, suprantigen, and concanavalin A despite normal T-cell receptor expression. Here, we show that the activation defect was due to a pleiotropic glycosylation abnormality because Dol-P-Man is required for both GPI anchor and N-linked oligosaccharide biosynthesis. When the yeast Dol-P-Man synthase gene was stably transfected into the mutants, full expression of surface GPI-anchored proteins was restored. However, N-linked glycosylation was either partially or completely corrected in different transfectants. Reconstitution of activation defects correlates well with the status of N-linked glycosylation, but not with the expression of GPI-anchored proteins. These results thus reveal an unexpected role of N-linked glycosylation in T-cell activation.

Insulin-Like Effects of Inositol Phosphate-Glycan on Messenger RNA Expression in Rat Hepatocytes

The ability of an inositol phosphate-glycan (IPG) to mimic the effects of insulin on regulation of the expression of specific mRNAs was studied in isolated hepatocytes from normal and diabetic rats. Incubation of normal liver cells with IPG (10 microM) during 90 min produced a 5-fold decrease in phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels, which had been previously increased about 10-fold by incubation with 8-bromo-cAMP (0.1 mM). The effect of IPG was dose dependent and could not be reproduced by galactose, glucosamine, or myo-inositol. IPG reduction of PEPCK mRNA is primarily due to a decrease in the rate of transcription of the gene, as judged by nuclear run-on transcription experiments performed in rat hepatoma H4IIE cells. In hepatocytes isolated from diabetic rats, treatment with 5 microM IPG for 15 min caused a 4-fold induction in the expression of alpha 2-microglobulin mRNA concomitantly with a 2.5-fold decrease in the level of PEPCK mRNA. Cleavage of IPG with nitrous acid abolished both the increase and the decrease in specific mRNAs levels. Glycosyl-phosphatidylinositol, the lipid precursor of IPG, did not modify either PEPCK or alpha 2-microglobulin mRNA levels. These data indicate that both positive and negative effects of insulin on the regulation of gene expression are mimicked by IPG.