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Dual Effect of the Adapter Growth Factor Receptor-Bound Protein 14 (Grb14) on Insulin Action in Primary Hepatocytes

Institut Cochin, Université Paris Descartes, Centre National de la Recherche Scientifique (Unité Mixte de Recherche 8104), 75014 Paris, France, and Institut National de la Santé et de la Recherche Médicale, Unité 567, 75014 Paris, France

Address all correspondence and requests for reprints to: Anne-Françoise Burnol, Institut Cochin, Département Endocrinologie, Métabolisme, Cancer, Faculté de Médecine Paris Descartes, 24 rue du Faubourg Saint-Jacques 75014 Paris, France. E-mail: burnol{at}cochin.inserm.fr.

	Abstract

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Tight control of insulin action in liver is a crucial determinant for the regulation of energy homeostasis. Growth factor receptor-bound protein 14 (Grb14) is a molecular adapter, highly expressed in liver, which binds to the activated insulin receptor and inhibits its tyrosine kinase activity. The physiological role of Grb14 in liver metabolism was unexplored. In this study we used RNA interference to investigate the consequences of Grb14 decrease on insulin-regulated intracellular signaling, and on glucose and lipid metabolism in mouse primary cultured hepatocytes. In Grb14-depleted hepatocytes, insulin-induced phosphorylation of Akt, and of its substrates glycogen synthase kinase 3 and fork-head box protein 1, was increased. These effects on insulin signaling are in agreement with the selective inhibitory effect of Grb14 on the receptor kinase. However, the metabolic and genic effects of insulin were differentially regulated after Grb14 down-regulation. Indeed, the insulin-mediated inhibition of hepatic glucose production and gluconeogenic gene expression was slightly increased. Surprisingly, despite the improved Akt pathway, the induction by insulin of sterol regulatory element binding protein-1c maturation was totally blunted. As a result, in the absence of Grb14, glycogen synthesis as well as glycolytic and lipogenic gene expression were not responsive to the stimulatory effect of insulin. This study provides evidence that Grb14 exerts a dual role on the regulation by insulin of hepatic metabolism. It inhibits insulin receptor catalytic activity, and acts also at a more distal step, i.e. sterol regulatory element binding protein-1c maturation, which effect is predominant under short-term inhibition of Grb14 expression.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
APPROPRIATE INSULIN ACTION on its target tissues is a crucial step to finely control glucose homeostasis. Alterations in the effects of insulin are associated with pathophysiological states such as obesity or type 2 diabetes, which are expanding in a worldwide epidemic way. The molecular mechanism underlying insulin resistance is not completely understood but is believed to involve impairments of the insulin receptor (IR) signaling network. The cascade of insulin signaling events is initiated by binding to its plasma membrane receptor, which induces IR autophosphorylation and activation of the tyrosine kinase domain, and leads to the recruitment and tyrosine phosphorylation of docking proteins such as insulin receptor substrates (IRSs) and Shc. These phosphorylated proteins then recruit other effectors through their SH2 domains, inducing the formation of macromolecular complexes and the activation of signaling cascades like the ERK1/2 pathway, and the phosphatidyl-inositol (PI) 3-kinase-Akt pathway. Akt is being viewed as a critical node in the metabolic actions elicited by insulin (1). Downstream the insulin signaling pathways, the transcription factor sterol regulatory element binding protein (SREBP)-1c has emerged as a major mediator of insulin action on hepatic glycolytic and lipogenic gene expression, and, therefore, as a key regulator of whole body energy metabolism (2). However, a tight regulation of insulin action is achieved by a subtle equilibrium between the activation of these signaling cascades and the simultaneous activation of counter-regulatory pathways.

Several feedback mechanisms have been implicated in the control of insulin signal transduction. Activation of phosphatases, like the protein tyrosine phosphatase 1B, which dephosphorylates and inactivates IR and IRS-1, or lipid phosphatases, like Src-homology inositol phosphatase 2 or phosphatase and tensin homolog, which counteract PI3-kinase activity, attenuated insulin signaling (3, 4, 5, 6). Different families of molecular adapters were also reported to inhibit insulin action, such as the Grb7 (growth factor receptor-bound protein 7) family of proteins, or suppressor of cytokine signaling (SOCS) proteins (for review, see Refs. 7 and 8). Implication of the SOCS proteins in insulin signaling and sensitivity has been more extensively studied, and it was shown that they act by disrupting insulin signaling through different molecular mechanisms, including inhibition of IR activity, competition for substrate recruitment and phosphorylation, and proteasomal degradation of the IRS proteins (9, 10, 11, 12, 13, 14). On the other hand, the Grb7 family of adapters comprises three proteins, Grb7, Grb10, and Grb14, but less information is available on their mechanism of action. Although the role of Grb7 in insulin signaling is unexplored, evidence is accumulating in favor of a role for Grb10 and Grb14 in insulin action. These proteins bind in vitro to the phosphorylated IR and inhibit its tyrosine kinase activity toward synthetic substrates (15). The crystal structure of the tyrosine kinase domain of the receptor in complex with the phosphorylated IR-interacting region between PH and SH2 (PIR-BPS) was recently resolved, and revealed that Grb14 acts as a pseudosubstrate inhibitor bound in the peptide binding groove of the kinase, and, thus, functions as a selective inhibitor of insulin signaling (16). Grb14 binding to the IR also modifies the interaction with protein tyrosine phosphatase 1B and regulates IR tyrosine phosphorylation in a site-specific manner (17). Overexpression of Grb10 or Grb14 in cultured cell lines decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and insulin distal effects such as DNA and glycogen synthesis (18, 19, 20, 21, 22). Importantly, both Grb14 and Grb10 deficient mice display an increased insulin sensitivity (23, 24, 25), suggesting that these proteins act indeed as inhibitors of the action of insulin in vivo.

Among the insulin target organs, the liver is a central player in the regulation of glucose and lipid metabolism. The crucial role of the IR in the regulation of glucose homeostasis and the maintenance of a normal hepatic function was highlighted by the phenotype of liver-specific IR knockout (LIRKO) mice, which exhibit a dramatic insulin resistance, with a failure of insulin to suppress hepatic glucose production and to regulate hepatic gene expression (26). Considering the inhibitory action of the Grb7 family of proteins on IR catalytic activity, it was of interest to investigate the physiological role of these adapters in the action of insulin in liver. Because Grb14 is highly expressed in liver and that Grb10 is virtually absent (24, 25, 27), we focused our studies on the role of Grb14. We used RNA interference to knock down the protein in primary cultured mouse hepatocytes, and studied insulin signaling and regulation of glucose and lipid metabolism.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Oligonucleotides and culture media were purchased from Invitrogen Corp. (Carlsbad, CA). Antibodies directed against the phosphorylated forms of ERK1/2 were from Promega Corp. (Madison, WI), and antibodies against the phosphorylated form of Akt (p-S473 and p-T308), glycogen synthase kinase (GSK) 3, and fork-head box protein (Foxo) 1 were obtained from Cell Signaling Technology, Inc. (Danvers, MA). Polyclonal antibodies against ERK1/2 and Akt were from Cell Signaling Technology. Polyclonal anti-Grb14 antibody was described previously (15), and fatty acid synthase (FAS) antibody was a gift from I. Dugail (Paris, France). All chemicals were from Sigma-Aldrich (St. Louis, MO).

Primary culture of hepatocytes and small interfering RNA (siRNA) transfection
Hepatocytes were isolated from livers of fed 8- to 10-wk-old male C57BL/6J mice by a modification of the collagenase method (28) and seeded as described previously (29). The studies were approved by the authors’ institutional committee on animal care. After cell attachment (4 h), the medium was replaced by fresh M199 medium containing 5 mM glucose, and supplemented with 100 µg/ml streptomycin, 100 U/ml penicillin, and 2.4 mM glutamine. The 21-nucleotide RNA with 3'-dTdT overhangs was synthesized by Dharmacon Research (Lafayette, CO). The siRNA sequence targeting mouse Grb14 (GenBank accession no. AF155647) was from position 396 relative to the start codon. The AA-N19 mRNA targets were 5'-CAGCTGTTGATCCTGAAGA-3' for Grb14 siRNA and 5'-ACGACGAGTAGTCTCTTGA-3' for scramble siRNA. Primary cultured hepatocytes were transiently transfected with siRNA duplexes using the technique described by Boussif et al. (30). Twenty-four hours after cell attachment, the hepatocytes were transfected with 200 pmol of either Grb14 or scramble siRNA and polyethylenimine in M199 medium in the presence of 5 mM glucose. To increase the efficiency of transfection, 300 plaque-forming units per cell of adenovirus Rous sarcoma virus promoter driving the nlsLacZ gene were added simultaneously to the hepatocytes (31). After transfection (5 h), the medium was changed, and transfected hepatocytes were cultured for 24 h in M199 medium containing 100 nM dexamethasone in the presence of 5 mM glucose or 25 mM glucose with or without insulin, as indicated. Dexamethasone was added to potentiate the effect of insulin, as described previously (32). Incubation in the presence of high glucose and insulin concentrations is likely to mimic conditions observed in the portal vein during the postprandial period, and was required to fully stimulate carbohydrate and lipid metabolism in primary cultured hepatocytes (29, 32).

Kinetics and Western blotting
After transfection, hepatocytes were cultured for 24 h in 25 mM glucose and then stimulated with or without 1 nM insulin for the indicated times. Cells were solubilized for 30 min at 4 C in lysis buffer and centrifuged at 15,000 x g for 15 min at 4 C as previously described (15). Protein concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA) with BSA as a standard. Equal amounts of proteins were subjected to SDS-PAGE analysis, and immunodetected with the indicated antibodies. Immunoreactive bands were revealed using the enhanced chemiluminescence detection kit (Pierce, Rockford, IL). Autoradiograms were scanned and quantified using an image processor program (Chemi Genius2 scan, GeneSnap; Syngene, Cambridge, UK).

Isolation of total RNA and analysis of their expression by real-time quantitative (RTQ) PCR
Twenty-four hours after siRNA transfection, hepatocytes were cultured for 24 h in the presence of the indicated concentrations of glucose and insulin, with the exception of the measurement of gluconeogenic gene expression. For phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) expression, 24 h after siRNA transfection, hepatocytes were cultured for 16 h in low glucose medium in the presence of 100 µM dibutyryl-cAMP and stimulated with the indicated concentrations of insulin during 6 h before RNA extraction. RNA was extracted using the RNeasy kit (QIAGEN, Inc., Valencia, CA). Total RNA was reverse transcribed for 1 h at 42 C using 100 U SuperScript II reverse transcriptase (Invitrogen). RTQ-PCR was performed with the LightCycler instrument (Roche Molecular Biochemicals, Indianapolis, IN) using SYBR Green I and the specific primers as described previously (27, 29, 33).

Hepatic glucose output
Twenty-four hours after siRNA transfection, hepatocytes were washed three times with warm PBS and incubated in M199 glucose free medium supplemented with lactate/pyruvate (10/1 mM) with or without 100 nM insulin. After 8-h incubation, cells were stimulated or not with 10 nM glucagon in the same medium. Incubation medium was collected after 16-h stimulation, and glucose production was measured by an enzymatic method (34).

Analysis of glycogen synthesis
Twenty-four hours after siRNA transfection, hepatocytes were cultured in M199 medium containing either 5 mM glucose or 25 mM glucose, supplemented or not with 1 or 100 nM insulin. After 24-h culture, the medium was replaced by the same fresh medium containing 2 µCi (for cells in 5 mM glucose) or 3 µCi (for cells in 25 mM glucose) of [¹⁴C]D-glucose (300 mCi/mmol/liter). The reaction was terminated after 3 h by washing the hepatocytes three times with ice-cold PBS and by the addition of 0.5 ml KOH 5N for cell solubilization. Cold glycogen carrier (2.5 mg) was added to the lysates, and samples were boiled for 30 min. Glycogen was precipitated overnight at –20 C by the addition of two volumes of cold ethanol 100%, and centrifuged at 10,000 x g for 10 min. Pellets were washed with 70% ethanol, resuspended in 0.5 ml water, and quantified by scintillation counting. Glycogen synthesis was expressed as nanomoles of glucose incorporated into glycogen in 3 h/mg proteins. For the determination of glycogen synthase (GS) activity, cells were snap frozen in liquid nitrogen and stored at –80 C until the extraction, as described in Ref. 35 . GS was determined in the whole homogenate from the incorporation of uridine 5'-diphosphate [6-³H]glucose into glycogen in the absence or presence of 10 mM glucose 6-phosphate for determination of active and total GS, respectively (36).

SREBP-1c precursor cleavage
Twenty-four hours after transfection, SREBP-1c expression was induced by the presence of 10 µM TO-901317 in the incubation medium for 6 h. Hepatocytes were treated or not with insulin (1 or 10 nM) for the final 30 min of this 6-h treatment period, as described (37).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Grb14 depletion induces an improvement of insulin-stimulated Akt pathway
Hepatocytes in primary cultures were transfected with 200 pmol of either Grb14 or scramble siRNA, and studied 24 h later. The Grb14 siRNA duplex used allowed a more than or equal to 80% inhibition of Grb14 expression, as shown in Fig. 1A. After gene silencing, hepatocytes were stimulated with 1 nM insulin for the indicated times, and the effect of Grb14 depletion on insulin-induced activation of the ERK1/2 and Akt pathways was studied. In hepatocytes either not transfected (data not shown) or treated with scramble siRNA, insulin caused a transient phosphorylation of ERK1/2 and a more sustained phosphorylation of Akt (Fig. 1, B and C). Grb14 knockdown led to a low but significant increase in ERK1 phosphorylation after 2 and 5-min insulin stimulation, and of ERK2 phosphorylation only after 2-min insulin. Ten minutes after the addition of insulin, ERK1 and ERK2 phosphorylation was similar in scramble siRNA and Grb14 siRNA treated hepatocytes. On the other hand, in Grb14-depleted cells, insulin-induced phosphorylation of both S473 and T308 residues of Akt was significantly increased for all times of insulin stimulation. One hour after the addition of insulin, S473 and T308 phosphorylation was respectively 2 and 2.5-fold higher in siGrb14-treated hepatocytes when compared with scramble siRNA. These differences were not due to variation in the amount of total Akt and ERK1/2 protein (Fig. 1, B and C, lower blots). Pretreatment of the hepatocytes with an inhibitor of PI3-kinase (LY294002) before insulin stimulation totally prevented Akt phosphorylation in hepatocytes, suggesting that the increase in Akt phosphorylation in the presence of siGrb14 was due to its upstream activation through the IR-PI3-kinase pathway (data not shown). The phosphorylation state of two substrates of Akt, GSK3 and Foxo1, was then analyzed. As shown in Fig. 1, D and E, after 3-h insulin stimulation, phosphorylation of these targets was enhanced in Grb14-depleted hepatocytes compared with scramble siRNA treated cells, confirming that the absence of Grb14 led to increased Akt activity. This increase in Akt substrate phosphorylation was still apparent in hepatocytes cultured for 24 h in the presence of insulin (Fig. 1, D and E).

View larger version (32K): [in this window] [in a new window]   FIG. 1. Grb14 knockdown enhances insulin (Ins) signaling. A, Efficiency of Grb14 silencing. Hepatocytes were transfected with the indicated siRNA and cultured in the presence of 25 mM glucose. Grb14 protein expression was analyzed 24 h later by Western blotting. β-Actin was immunodetected in the same extracts as a control. B and C, Twenty-four hours after siRNA transfection, hepatocytes cultured in the presence of 25 mM glucose were treated with 1 nM insulin for the indicated times. Whole cell lysates were analyzed by SDS-PAGE, and immunodetected with antibodies against the total and phosphorylated forms of Akt and ERK1/2 as indicated. The Western blots shown are representative of five to seven independent experiments. The autoradiograms were quantified by scanning densitometry, and the means ± SE are represented as histograms. C, Results are expressed as percentage of the value at 2 min. Because variations of p-T308 and p-S473 for scramble siRNA (white bars) transfected hepatocytes were similar, for clarity of representation, they are presented as a single column. D and E, Twenty-four hours after siRNA transfection, hepatocytes were cultured in the presence of the indicated concentrations of glucose [5 mM glucose (G5) or 25 mM glucose (G25)] and insulin (0, 1, or 100 nM) for the indicated time (3 or 24 h). Whole cell lysates were analyzed by SDS-PAGE, and immunodetected with antibodies against the total or phosphorylated forms of GSK3 and the phosphorylated form of Foxo1. The Western blots shown are representative of four independent experiments. The autoradiograms were quantified by scanning densitometry, and the means ± SE are represented as histograms. Black bars indicate Grb14 siRNA. Statistical significance was established, and differences are represented as follows: *, P < 0.05 and **, P < 0.01 for Grb14 siRNA compared with scramble siRNA treated hepatocytes in the same conditions of culture; #, P < 0.05 and ##, P < 0.01 for the effect of insulin in scramble siRNA treated hepatocytes; and , P < 0.05 and , P < 0.01 for the effect of insulin in Grb14 siRNA treated hepatocytes.

Consequences of Grb14 depletion insulin regulation of glucose and lipid metabolism
To determine whether the increased activation of an early insulin signaling step in Grb14-depleted hepatocytes induced an improvement in glucose metabolism, we first measured insulin-induced suppression of hepatic glucose production. Glucagon induced a similar increase in glucose output in both scramble and Grb14 siRNA transfected hepatocytes. The addition of insulin decreased glucagon-induced hepatic glucose production, and this effect was slightly but significantly increased in Grb14 siRNA treated hepatocytes compared with control scramble siRNA treated cells (Fig. 2A). This was correlated with an enhanced inhibitory action of insulin on PEPCK mRNA expression, but not of G-6-Pase expression, in Grb14-depleted hepatocytes (Fig. 2, B and C). These data suggest that the inhibitory action of insulin on hepatic glucose output was slightly but significantly enhanced in the absence of Grb14.

View larger version (29K): [in this window] [in a new window]   FIG. 2. Effect of Grb14 gene silencing on the gluconeogenic pathway and leptin receptor expression. A, Hepatic glucose production. Twenty-four hours after siRNA transfection, hepatocytes were cultured in the absence of glucose with the addition of glucagon to induce the gluconeogenic pathway as described in the Materials and Methods. B and C, Expression of gluconeogenic enzymes. After siRNA transfection hepatocytes were cultured in medium with 5 mM glucose supplemented with dibutyryl-cAMP as described in the Materials and Methods. PEPCK (B) and G-6-Pase (C) were measured by RTQ-PCR. D, Expression of the leptin receptor ObRa. Twenty-four hours after siRNA transfection, hepatocytes were cultured for 24 h in the presence of the indicated concentrations of glucose [5 mM glucose (G5) or 25 mM glucose (G25)] and insulin (Ins). ObRa expression was measured by RTQ-PCR. Gene expression was normalized to cyclophilin mRNA values. The results shown are the mean ± SE of three to four independent experiments. Black bars indicate Grb14 siRNA and white bars scramble siRNA. Statistical significance was established and represented as follows: #, P < 0.05 and ##, P < 0.01 for the effect of insulin in scramble siRNA treated hepatocytes; , P < 0.05 and , P < 0.01 for the effect of insulin in Grb14 siRNA treated hepatocytes; and *, P < 0.05 and **, P < 0.01 for Grb14 siRNA compared with scramble siRNA treated hepatocytes in the same conditions of culture.

We next investigated the effect of insulin on glycogen synthesis. In scramble siRNA transfected hepatocytes, insulin induced an increase of glucose incorporation into glycogen in a dose-dependent manner. However, unexpectedly, the effect of insulin on glycogen synthesis was totally prevented in Grb14 siRNA treated hepatocytes (Fig. 3A). This brought us to investigate the mechanisms leading to this defect, and to measure GS activity. Insulin induced a dose-dependent increase in GS activity in scramble siRNA treated hepatocytes, whereas it was unable to enhance GS activity in Grb14-depleted cells (Fig. 3B). On the other hand, glycogen phosphorylase activity was similar in control and Grb14-depleted hepatocytes (data not shown). GS activity is controlled by various mechanisms, including its inhibition by GSK3 phosphorylation and its allosteric activation by glucose-6-phosphate (38). As shown in Fig. 1D, insulin-mediated phosphorylation of GSK3 was higher in Grb14 siRNA transfected hepatocytes, suggesting that it was less active, and that modification in GSK3 activity cannot account for the inhibition of GS activity. In hepatocytes, glucose is phosphorylated into glucose-6-phosphate by glucokinase (GK), whose expression is strictly dependent on insulin action (39). As shown in Fig. 3C, GK expression was stimulated by insulin in scramble siRNA transfected hepatocytes, but this stimulation was completely prevented by Grb14 knockdown. The decrease in GK expression under insulin stimulation in Grb14-depleted hepatocytes could at least partly explain the lack of effect of insulin on glucose incorporation into glycogen.

View larger version (29K): [in this window] [in a new window]   FIG. 3. Grb14 knockdown inhibits insulin (Ins) stimulation of glycogen synthesis and glycolytic and lipogenic pathways. Twenty-four hours after siRNA transfection, hepatocytes were cultured for 24 h in the presence of the indicated concentrations of glucose [5 mM glucose (G5) or 25 mM glucose (G25)] and insulin. Glycogen synthesis (A) and GS activity (B) were measured as described in the Materials and Methods. C and D, GK and FAS gene expression were measured by RTQ-PCR. Gene expression was normalized to cyclophilin mRNA values. E, FAS protein expression. Whole cell lysates were analyzed by SDS-PAGE and immunodetected with an antibody against FAS (left). The autoradiograms were quantified by scanning densitometry, and the means ± SE are represented as histograms (right). The results shown are the mean ± SE of three (A and B), four (C and E), or seven (D) independent experiments. Black bars indicate Grb14 siRNA and white bars scramble siRNA. Statistical significance was established, and differences are represented as follows: *, P < 0.05 and **, P < 0.01 for Grb14 siRNA compared with scramble siRNA treated hepatocytes in the same conditions of culture; #, P < 0.05 and ##, P < 0.01 for the effect of insulin in scramble siRNA treated hepatocytes; and , P < 0.05 and , P < 0.01 for the effect of insulin in Grb14 siRNA treated hepatocytes.

SREBP-1c expression and maturation is altered in Grb14-depleted hepatocytes
The stimulatory effect of insulin on the expression of glycolytic and lipogenic genes is mediated by the transcriptional factor SREBP-1c. SREBP-1c expression itself is stimulated by insulin both at the transcriptional and the posttranslational levels. Insulin induces SREBP-1c transcription through a sterol-responsive element sequence in its promoter, suggesting that SREBP-1c controls its own expression (41). SREBP-1c is synthesized as a precursor form anchored in the endoplasmic reticulum (ER), and after translocation in the Golgi apparatus, is cleaved by a proteolytic process regulated by insulin (37). The mature active form can then be translocated into the nucleus to stimulate transcription of its target genes. We first determined whether the induction by insulin of SREBP-1c expression was altered by Grb14 knockdown. Insulin induced a 1.8-fold increase in SREBP-1c mRNA in control hepatocytes but had no effect in Grb14-depleted hepatocytes (Fig. 4A). Therefore, we investigated the effect of Grb14 depletion on the ability of insulin to stimulate the proteolytic processing of SREBP-1c. To induce an insulin-independent SREBP-1c precursor expression, siRNA transfected hepatocytes were incubated in the presence of TO-901317, a liver X receptor agonist known to robustly stimulate SREBP-1c precursor accumulation in the ER, and then insulin was added for the last 30 min (37). This short-time insulin incubation has no effect on SREBP-1c mRNA expression but is able to increase the presence of mature SREBP-1c in the nucleus. Synthesis of SREBP-1c precursor was similar in scramble and Grb14 siRNA transfected hepatocytes (Fig. 4B). The addition of insulin increased significantly the accumulation of the mature cleaved form of SREBP-1c in the nucleus of control hepatocytes, as previously described (37), but was totally inefficient in Grb14-depleted cells. These results show that in the absence of Grb14, insulin is unable to stimulate SREBP-1c maturation.

View larger version (20K): [in this window] [in a new window]   FIG. 4. Decreased effect of insulin (Ins) on SREBP-1c expression and maturation after Grb14 knockdown. A, C, and D, SREBP-1c, Insig2a, and Insig1 gene expression, respectively. Hepatocytes were cultured as described in Fig. 2D. Gene expression was measured by RTQ-PCR and normalized to cyclophilin mRNA values. Results are the mean ± SE of four to seven independent experiments. B, Insulin-induced SREBP-1c cleavage. After siRNA transfection hepatocytes were cultured for 24 h in 5 mM glucose with the addition of TO-901317 during the last 6 h to induce SREBP-1c protein expression, and were stimulated or not with insulin (1 or 10 nM) for the final 30 min. The Western blot shown is representative of four independent experiments. The autoradiograms were quantified by scanning densitometry, and the means ± SE are represented as histograms. Black bars indicate Grb14 siRNA and white bars scramble siRNA. Statistical significance was established and represented as follows: #, P < 0.05 and ##, P < 0.01 for the effect of insulin compared with glucose 25 mM (G25) in scramble siRNA treated hepatocytes for A, B, and D, and with glucose 5 mM (G5) for C; , P < 0.05 and , P < 0.01 for the effect of insulin compared with glucose 25 mM in Grb14 siRNA treated hepatocytes for A, B, and D, and with glucose 5 mM for B; and *, P < 0.05 and **, P < 0.01 for Grb14 siRNA compared with scramble siRNA treated hepatocytes in the same conditions of culture. mSREBP-1c, Mature SREBP-1c; pSREBP-1c, SREBP-1c precursor.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The molecular adapter Grb14 was identified as an interacting partner of the autophosphorylated IR, specifically expressed in insulin-sensitive tissues (19). As demonstrated by in vitro experiments using purified proteins, and by the crystal structure of the complex between the tyrosine kinase domain of the IR and the Grb14 PIR-BPS domain, Grb14 acts as a selective inhibitor of IR catalytic activity (15, 16). Consistent with this inhibitory action, Grb14 gene KO mice displayed an enhanced insulin signaling, but this phenotype was apparent only in adult male Grb14 KO mice over 16–20 wk of age (23). On the other hand, the phenotype of KO mice could also be the result of compensatory mechanisms that take place during the development and growth of the mice. Thus, to evaluate the physiological importance of this protein in the liver and study the consequences of an acute decrease in Grb14 expression, we used siRNA in primary cultured hepatocytes. The present experiments show that, as anticipated, the early steps of insulin signaling pathways were improved by Grb14 knockdown. However, the distal steps of the effects of insulin on metabolic pathways were differentially altered (Fig. 5). Similar results were obtained using another sequence for RNA interference (data not shown), suggesting that the results obtained cannot be attributed to an unspecific action of the siRNA used. The paradoxical consequences of Grb14 depletion were unexpected, and suggested that Grb14 action was not limited to its role as a direct inhibitor of the IR catalytic activity, and that under an acute modulation of its level of expression, a more distal effect of Grb14 was predominant.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Schematic representation of the consequences of RNA interference-induced decrease in Grb14 expression on insulin signaling in hepatocytes. A decrease in the Grb14 inhibitory effect on IR catalytic activity resulted in an increased Akt phosphorylation and activity. The inhibition of insulin-induced SREBP-1c maturation and activation of target genes provided evidence for a distal role of Grb14 downstream from Akt.

A major finding of the present study was that knock down of Grb14 induced a decrease in a number of the effects of insulin, such as expression of SREBP-1c, Insig1, GK, and FAS, and glycogen synthesis. The protein kinase (PK) A signaling pathway or SOCS3 expression was not altered (data not shown), suggesting that a compensatory increase in insulin counter-regulatory pathways was not implicated in the modifications induced by Grb14 depletion. Interestingly, an upstream defect in insulin-induced accumulation of nuclear SREBP-1c could be a simple explanation for all these observations. Indeed, a decrease in insulin effect on SREBP-1c maturation should inhibit insulin stimulation of SREBP-1c expression and, consecutively, the transcriptional activation of its target genes. The reduced expression of GK could then explain the lack of effect of insulin on glycogen synthesis in Grb14-depleted hepatocytes. The acute effect of insulin to stimulate SREBP-1c precursor maturation occurs via a PI3-kinase dependent mechanism (37). A possible explanation for the paradoxical inhibition of insulin-induced SREBP-1c maturation in the absence of Grb14, despite an improved PI3-kinase-Akt pathway, could be that in addition to its inhibitory action on IR catalytic activity, the molecular adapter Grb14 interfered with insulin signaling at a step downstream of Akt activation, and interacted with an unidentified partner involved in insulin stimulation of SREBP-1c nuclear accumulation. SREBP activity and expression of the SREBP-responsive genes can be regulated either by the cleavage of the precursor and by the stability of the nuclear form that is controlled by the ubiquitin-proteasome pathway (45, 46). Thus, we cannot exclude that the absence of accumulation of mature SREBP-1c in the nucleus in response to insulin in Grb14-depleted hepatocytes could also be attributed to an increased degradation of the active form.

The molecular mechanism involved in the acute stimulation by insulin of the proteolytic processing of SREBP-1c is still unclear. It has been proposed that insulin-mediated down-regulation of Insig2a may promote the cleavage of SREBP-1c (44). However, there is no direct relationship between the expression level of Insig and the efficiency of insulin-induced SREBP-1c cleavage. Indeed, insulin stimulated SREBP-1c maturation in the presence of high levels of Insig2a and Insig1 (37), and we report in the present study an inhibition of the cleavage induced by insulin despite low levels of expression of Insig. This suggests that, in addition to the expression of its anchoring proteins, other mechanisms are important for SREBP-1c maturation. On the other hand, recent studies described the regulation of the export of the Scap-SREBP complex from the ER to the Golgi by cholesterol binding to Scap or oxysterol binding to Insig, and showed that it is a major regulatory step in SREBP cleavage (47, 48). By analogy to this mechanism, one can propose that Grb14 interacts with a protein involved in the formation of a complex between Insig and Scap, or in the recognition of the sorting signal of Scap in response to insulin.

As a molecular adapter, Grb14 is composed of a succession of protein-protein interacting domains (49). Furthermore, in vivo insulin stimulation induces the recruitment of only a small fraction of hepatic Grb14 to the IR present at the plasma membrane (Desbuquois, B., and A.-F. Burnol, unpublished data), suggesting that in addition to its inhibitory effect at the level of the receptor, Grb14 must also act by its interaction with other partners. At the present time, only a few Grb14 partners have been described (49). Grb14 interacts constitutively with phosphoinositide-dependent kinase (PDK)-1, an upstream kinase activating Akt in response to insulin stimulation. The Grb14-PDK-1 association is required to recruit PDK-1 to the activated IR, and to promote Akt activation in human embryonic kidney transfected cells (50). However, Akt phosphorylation and activation were significantly increased in Grb14-depleted hepatocytes, giving evidence that disruption of the Grb14-PDK-1 complex could not explain the inhibition of SREBP-1c maturation observed. Grb14 also interacts with the PKC -interacting protein (ZIP). ZIP favors Grb14 phosphorylation by PKC, and enhances its inhibitory effect on IR kinase activity (51). ZIP is able to bind to both PKC and PKC, two atypical PKCs similarly expressed in liver (52, 53). It was recently documented that the PKC/ pathway was important for the up-regulation of SREBP-1c expression in response to insulin (53, 54). In mice specifically deprived of PKC in liver, the induction of SREBP-1c expression was decreased, whereas the inhibitory effect of insulin on gluconeogenic gene expression was preserved, or slightly increased (53), which is similar to the alterations induced by Grb14 depletion in hepatocytes reported in the present study. A discrepancy between these two models is that insulin normally stimulated GK expression and glycogen synthesis in the absence of liver PKC, whereas these pathways were totally blunted in the absence of Grb14. This suggests that disruption of the Grb14-ZIP interaction in Grb14-depleted hepatocytes can be involved in the modifications observed in insulin effects by altering atypical PKC signaling, but Grb14 regulation of the metabolic actions of insulin in the liver should involve additional mechanisms.

In conclusion, the present study provides evidence that, in addition to its inhibition of IR catalytic activity, Grb14 regulates insulin action in hepatocytes by acting at a more distal step, which is predominant under a short-term modulation of Grb14 expression. Interestingly, Grb14 depletion maintains the effect of insulin on hepatic glucose production while inhibiting the lipogenic pathway, suggesting that regulation of the Grb14 level of expression and action could represent a potential method to modulate the metabolic actions of insulin.

	Acknowledgments

 
We thank Dr. L. Agius for providing help with the measurement of glycogen synthase activity, and I. Dugail for the fatty acid synthase antibody. We also thank Catherine Postic and Pascale Bossard for critically reading the manuscript.

	Footnotes

 
This work was supported by a grant from the Ministère de l'Enseignement Supérieur et la Recherche (Action Concertée Incitative no. 02 2 0537/8). N.C. is the recipient of a doctoral fellowship from the Ministère de l'Enseignement Supérieur et la Recherche and received financial support from Alfediam/Roche Diagnostics.

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 27, 2008

Abbreviations: ER, Endoplasmic reticulum; FAS, fatty acid synthase; Foxo, fork-head box protein; G-6-Pase, glucose-6-phosphatase; GK, glucokinase; Grb, growth factor receptor-bound protein; GS, glycogen synthase; GSK, glycogen synthase kinase; Insig, insulin-induced gene; IR, insulin receptor; IRS, insulin receptor substrate; KO, knockout; LIRKO, liver-specific IR knockout; ObRa, leptin receptor; PDK, phosphoinositide-dependent kinase; PEPCK, phosphoenolpyruvate carboxykinase; PI, phosphatidyl-inositol; PK, protein kinase; RTQ, real-time quantitative; Scap, sterol regulatory element binding protein cleavage-activating protein; siRNA, small interfering RNA; SOCS, suppressor of cytokine signaling; SREBP, sterol regulatory element binding protein; ZIP, protein kinase C -interacting protein.

Received August 29, 2007.

Accepted for publication February 29, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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