Search

Liver physiology is circadian and sensitive to feeding and insulin. Food intake regulates insulin secretion and is a dominant signal for the liver clock. However, how much insulin contributes to the effect of feeding on the liver clock and rhythmic gene expression remains to be investigated. Insulin action partly depends on changes in insulin receptor (IR)-dependent gene expression. Here, we use hepatocyte-restricted gene deletion of IR to evaluate its role in the regulation and oscillation of gene expression as well as in the programming of the circadian clock in the adult mouse liver. We find that, in the absence of IR, the rhythmicity of core-clock gene expression is altered in response to day-restricted feeding. This change in core-clock gene expression is associated with defective reprogramming of liver gene expression. Our data show that an intact hepatocyte insulin receptor is required to program the liver clock and associated rhythmic gene expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: A common feature of metabolic diseases is their association with chronic low-grade inflammation. While enhanced gut permeability and systemic bacterial endotoxin translocation have been suggested as key players of this metaflammation, the mechanistic bases underlying these features upon the diabesity cascade remain partly understood., Methods: Here, we show in mice that, independently of obesity, the induction of acute and global insulin resistance and associated hyperglycemia, upon treatment with an insulin receptor (IR) antagonist (S961), elicits gut hyperpermeability without triggering systemic inflammatory response., Results: Of note, S961-treated diabetic mice display major defects of gut barrier epithelial functions, such as increased epithelial paracellular permeability and impaired cell-cell junction integrity. We also observed in these mice the early onset of a severe gut dysbiosis, as characterized by the bloom of pro-inflammatory Proteobacteria, and the later collapse of Paneth cells antimicrobial defense. Interestingly, S961 treatment discontinuation is sufficient to promptly restore both the gut microbial balance and the intestinal barrier integrity. Moreover, fecal transplant approaches further confirm that S961-mediated dybiosis contributes at least partly to the disruption of the gut selective epithelial permeability upon diabetic states., Conclusions: Together, our results highlight that insulin signaling is an indispensable gatekeeper of intestinal barrier integrity, acting as a safeguard against microbial imbalance and acute infections by enteropathogens., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Objective: Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of pancreatic β-cells producing insulin. Both T1D patients and animal models exhibit gut microbiota and mucosa alterations, although the exact cause for these remains poorly understood. We investigated the production of key cytokines controlling gut integrity, the abundance of segmented filamentous bacteria (SFB) involved in the production of these cytokines, and the respective role of autoimmune inflammation and hyperglycaemia., Design: We used several mouse models of autoimmune T1D as well as mice rendered hyperglycaemic without inflammation to study gut mucosa and microbiota dysbiosis. We analysed cytokine expression in immune cells, epithelial cell function, SFB abundance and microbiota composition by 16S sequencing. We assessed the role of anti-tumour necrosis factor α on gut mucosa inflammation and T1D onset., Results: We show in models of autoimmune T1D a conserved loss of interleukin (IL)-17A, IL-22 and IL-23A in gut mucosa. Intestinal epithelial cell function was altered and gut integrity was impaired. These defects were associated with dysbiosis including progressive loss of SFB. Transfer of diabetogenic T-cells recapitulated these gut alterations, whereas induction of hyperglycaemia with no inflammation failed to do so. Moreover, anti-inflammatory treatment restored gut mucosa and immune cell function and dampened diabetes incidence., Conclusion: Our results demonstrate that gut mucosa alterations and dysbiosis in T1D are primarily linked to inflammation rather than hyperglycaemia. Anti-inflammatory treatment preserves gut homeostasis and protective commensal flora reducing T1D incidence., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

TxNIP (Thioredoxin-interacting protein) is considered as a potential drug target for type 2 diabetes. Although TxNIP expression is correlated with hyperglycemia and glucotoxicity in pancreatic β cells, its regulation in liver cells has been less investigated. In the current study, we aim at providing a better understanding of Txnip regulation in hepatocytes in response to physiological stimuli and in the context of hyperglycemia in db/db mice. We focused on regulatory pathways governed by ChREBP (Carbohydrate Responsive Element Binding Protein) and FoxO1 (Forkhead box protein O1), transcription factors that play central roles in mediating the effects of glucose and fasting on gene expression, respectively. Studies using genetically modified mice reveal that hepatic TxNIP is up-regulated by both ChREBP and FoxO1 in liver cells and that its expression strongly correlates with fasting, suggesting a major role for this protein in the physiological adaptation to nutrient restriction., Competing Interests: The authors declare no conflict of interest., (© 2021.)

Aim: In hepatocytes, the peroxisome proliferator-activated receptor (PPAR)-α and insulin receptor (IR) are critical for transcriptional responses to fasting and feeding, respectively. The present report analyzes the effects of nutritional status (fasting vs feeding) on the expression of a large panel of hepatokines in hepatocyte-specific PPAR-α (Pparα hep-/- ) and IR (IR hep-/- ) null mice., Methods: Pparα hep-/- and IR hep-/- mice, and their wild-type littermates, were subjected to fasting or feeding metabolic challenges, then analyzed for hepatokine gene expression. Experiments were conducted in mice of both genders., Results: Our data confirmed that PPAR-α is essential for regulating fasting-induced Fgf21 and Angptl4 expression. In mice lacking PPAR-α, fasting led to increased Igfbp1 and Gdf15 gene expression. In the absence of hepatic IR, feeding induced overexpression of Igfbp1, follistatin (Fst) and adropin (Enho), and reduced activin E (Inhbe) expression. Gender had only a modest influence on hepatokine gene expression in the liver., Conclusion: The present results highlight the potential roles of hepatokines as a class of hormones that substantially influence nutritional regulation in both female and male mice., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

The Wnt/β-catenin pathway plays a pivotal role in liver structural and metabolic homeostasis. Wnt activity is tightly regulated by the acyltransferase Porcupine through the addition of palmitoleate. Interestingly palmitoleate can be endogenously produced by the stearoyl-CoA desaturase 1 (SCD1), a lipogenic enzyme transcriptionally regulated by insulin. This study aimed to determine whether nutritional conditions, and insulin, regulate Wnt pathway activity in liver. An adenoviral TRE-Luciferase reporter was used as a readout of Wnt/β-catenin pathway activity, in vivo in mouse liver and in vitro in primary hepatocytes. Refeeding enhanced TRE-Luciferase activity and expression of Wnt target genes in mice liver, revealing a nutritional regulation of the Wnt/β-catenin pathway. This effect was inhibited in liver specific insulin receptor KO (iLIRKO) mice and upon wortmannin or rapamycin treatment. Overexpression or inhibition of SCD1 expression regulated Wnt/β-catenin activity in primary hepatocytes. Similarly, palmitoleate added exogenously or produced by SCD1-mediated desaturation of palmitate, induced Wnt signaling activity. Interestingly, this effect was abolished in the absence of Porcupine, suggesting that both SCD1 and Porcupine are key mediators of insulin-induced Wnt/β-catenin activity in hepatocytes. Altogether, our findings suggest that insulin and lipogenesis act as potential novel physiological inducers of hepatic Wnt/β-catenin pathway.

Thioredoxin interacting protein (TxNIP), which strongly responds to glucose, has emerged as a central mediator of glucotoxicity in pancreatic β cells. TxNIP is a scaffold protein interacting with target proteins to inhibit or stimulate their activity. Recent studies reported that high glucose stimulates the interaction of TxNIP with the inflammasome protein NLRP3 (NLR family, pyrin domain containing 3) to increase interleukin-1 β (IL1β) secretion by pancreatic β cells. To better understand the regulation of TxNIP by glucose in pancreatic β cells, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAcylation) in regulating TxNIP at the posttranslational level. O-GlcNAcylation of proteins is controlled by two enzymes: the O-GlcNAc transferase (OGT), which transfers a monosaccharide to serine/threonine residues on target proteins, and the O-GlcNAcase (OGA), which removes it. Our study shows that TxNIP is subjected to O-GlcNAcylation in response to high glucose concentrations in β cell lines. Modification of the O-GlcNAcylation pathway through manipulation of OGT or OGA expression or activity significantly modulates TxNIP O-GlcNAcylation in INS1 832/13 cells. Interestingly, expression and O-GlcNAcylation of TxNIP appeared to be increased in islets of diabetic rodents. At the mechanistic level, the induction of the O-GlcNAcylation pathway in human and rat islets promotes inflammasome activation as evidenced by enhanced cleaved IL1β. Overexpression of OGT in HEK293 or INS1 832/13 cells stimulates TxNIP and NLRP3 interaction, while reducing TxNIP O-GlcNAcylation through OGA overexpression destabilizes this interaction. Altogether, our study reveals that O-GlcNAcylation represents an important regulatory mechanism for TxNIP activity in β cells.

Metabolic diseases are characterized by a decreased action of insulin. During the course of the disease, usual treatments frequently fail and patients are finally submitted to insulinotherapy. There is thus a need for innovative therapeutic strategies to improve insulin action. Growth factor receptor-bound protein 14 (Grb14) is a molecular adapter that specifically binds to the activated insulin receptor (IR) and inhibits its tyrosine kinase activity. Molecules disrupting Grb14-IR binding are therefore potential insulin-sensitizing agents. We used Structure-Based Virtual Ligand Screening to generate a list of 1000 molecules predicted to hinder Grb14-IR binding. Using an acellular bioluminescence resonance energy transfer (BRET) assay, we identified, out of these 1000 molecules, 3 compounds that inhibited Grb14-IR interaction. Their inhibitory effect on insulin-induced Grb14-IR interaction was confirmed in co-immunoprecipitation experiments. The more efficient molecule (C8) was further characterized. C8 increased downstream Ras-Raf and PI3-kinase insulin signaling, as shown by BRET experiments in living cells. Moreover, C8 regulated the expression of insulin target genes in mouse primary hepatocytes. These results indicate that C8, by reducing Grb14-IR interaction, increases insulin signalling. The use of C8 as a lead compound should allow for the development of new molecules of potential therapeutic interest for the treatment of diabetes.

While the physiological benefits of the fibroblast growth factor 21 (FGF21) hepatokine are documented in response to fasting, little information is available on Fgf21 regulation in a glucose-overload context. We report that peroxisome-proliferator-activated receptor α (PPARα), a nuclear receptor of the fasting response, is required with the carbohydrate-sensitive transcription factor carbohydrate-responsive element-binding protein (ChREBP) to balance FGF21 glucose response. Microarray analysis indicated that only a few hepatic genes respond to fasting and glucose similarly to Fgf21. Glucose-challenged Chrebp -/- mice exhibit a marked reduction in FGF21 production, a decrease that was rescued by re-expression of an active ChREBP isoform in the liver of Chrebp -/- mice. Unexpectedly, carbohydrate challenge of hepatic Pparα knockout mice also demonstrated a PPARα-dependent glucose response for Fgf21 that was associated with an increased sucrose preference. This blunted response was due to decreased Fgf21 promoter accessibility and diminished ChREBP binding onto Fgf21 carbohydrate-responsive element (ChoRE) in hepatocytes lacking PPARα. Our study reports that PPARα is required for the ChREBP-induced glucose response of FGF21., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Identification of the Mondo glucose-responsive transcription factors family, including the MondoA and MondoB/ChREBP paralogs, has shed light on the mechanism whereby glucose affects gene transcription. They have clearly emerged, in recent years, as key mediators of glucose sensing by multiple cell types. MondoA and ChREBP have overlapping yet distinct expression profiles, which underlie their downstream targets and separate roles in regulating genes involved in glucose metabolism. MondoA can restrict glucose uptake and influences energy utilization in skeletal muscle, while ChREBP signals energy storage through de novo lipogenesis in liver and white adipose tissue. Because Mondo proteins mediate metabolic adaptations to changing glucose levels, a better understanding of cellular glucose sensing through Mondo proteins will likely uncover new therapeutic opportunities in the context of the imbalanced glucose homeostasis that accompanies metabolic diseases such as type 2 diabetes and cancer. Here, we provide an overview of structural homologies, transcriptional partners as well as the nutrient and hormonal mechanisms underlying Mondo proteins regulation. We next summarize their relative contribution to energy metabolism changes in physiological states and the evolutionary conservation of these pathways. Finally, we discuss their possible targeting in human pathologies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Metabolic diseases such as obesity and type 2 diabetes are recognized as independent risk factors for hepatocellular carcinoma (HCC). Hyperinsulinemia, a hallmark of these pathologies, is suspected to be involved in HCC development. The molecular adapter growth factor receptor binding protein 14 (Grb14) is an inhibitor of insulin receptor catalytic activity, highly expressed in the liver. To study its involvement in hepatocyte proliferation, we specifically inhibited its liver expression using a short hairpin RNA strategy in mice. Enhanced insulin signaling upon Grb14 inhibition was accompanied by a transient induction of S-phase entrance by quiescent hepatocytes, indicating that Grb14 is a potent repressor of cell division. The proliferation of Grb14-deficient hepatocytes was cell-autonomous as it was also observed in primary cell cultures. Combined Grb14 down-regulation and insulin signaling blockade using pharmacological approaches as well as genetic mouse models demonstrated that Grb14 inhibition-mediated hepatocyte division involved insulin receptor activation and was mediated by the mechanistic target of rapamycin complex 1-S6K pathway and the transcription factor E2F1. In order to determine a potential dysregulation in GRB14 gene expression in human pathophysiology, a collection of 85 human HCCs was investigated. This revealed a highly significant and frequent decrease in GRB14 expression in hepatic tumors when compared to adjacent nontumoral parenchyma, with 60% of the tumors exhibiting a reduced Grb14 mRNA level., Conclusion: Our study establishes Grb14 as a physiological repressor of insulin mitogenic action in the liver and further supports that dysregulation of insulin signaling is associated with HCC. (Hepatology 2017;65:1352-1368)., (© 2016 by the American Association for the Study of Liver Diseases.)

A long-standing paradox in the pathophysiology of metabolic diseases is the selective insulin resistance of the liver. It is characterized by a blunted action of insulin to reduce glucose production, contributing to hyperglycemia, while de novo lipogenesis remains insulin sensitive, participating in turn to hepatic steatosis onset. The underlying molecular bases of this conundrum are not yet fully understood. Here, we established a model of selective insulin resistance in mice by silencing an inhibitor of insulin receptor catalytic activity, the growth factor receptor binding protein 14 (Grb14) in liver. Indeed, Grb14 knockdown enhanced hepatic insulin signaling but also dramatically inhibited de novo fatty acid synthesis. In the liver of obese and insulin-resistant mice, downregulation of Grb14 markedly decreased blood glucose and improved liver steatosis. Mechanistic analyses showed that upon Grb14 knockdown, the release of p62/sqstm1, a partner of Grb14, activated the transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2), which in turn repressed the lipogenic nuclear liver X receptor (LXR). Our study reveals that Grb14 acts as a new signaling node that regulates lipogenesis and modulates insulin sensitivity in the liver by acting at a crossroad between the insulin receptor and the p62-Nrf2-LXR signaling pathways., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Defective hepatic insulin receptor (IR) signalling is a pathogenic manifestation of metabolic disorders including obesity and diabetes. The endo/lysosomal trafficking system may coordinate insulin action and nutrient homeostasis by endocytosis of IR and the autophagic control of intracellular nutrient levels. Here we show that class III PI3K--a master regulator of endocytosis, endosomal sorting and autophagy--provides negative feedback on hepatic insulin signalling. The ultraviolet radiation resistance-associated gene protein (UVRAG)-associated class III PI3K complex interacts with IR and is stimulated by insulin treatment. Acute and chronic depletion of hepatic Vps15, the regulatory subunit of class III PI3K, increases insulin sensitivity and Akt signalling, an effect that requires functional IR. This is reflected by FoxO1-dependent transcriptional defects and blunted gluconeogenesis in Vps15 mutant cells. On depletion of Vps15, the metabolic syndrome in genetic and diet-induced models of insulin resistance and diabetes is alleviated. Thus, feedback regulation of IR trafficking and function by class III PI3K may be a therapeutic target in metabolic conditions of insulin resistance.

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Beyond its key role in the control of energy metabolism, insulin is also an important regulator of cell division and neoplasia. However, the molecular events involved in insulin-driven cell proliferation are not fully elucidated. Here, we show that the ubiquitin ligase Chfr, a checkpoint protein involved in G2/M transition, is a new effector involved in the control of insulin-induced cell proliferation. Chfr is identified as a partner of the molecular adapter Grb14, an inhibitor of insulin signalling. Using mammalian cell lines and the Xenopus oocyte as a model of G2/M transition, we demonstrate that Chfr potentiates the inhibitory effect of Grb14 on insulin-induced cell division. Insulin stimulates Chfr binding to the T220 residue of Grb14. Both Chfr binding site and Grb14 C-ter BPS-SH2 domain, mediating IR binding and inhibition, are required to prevent insulin-induced cell division. Targeted mutagenesis revealed that Chfr ligase activity and phosphorylation of its T39 residue, a target of Akt, are required to potentiate Grb14 inhibitory activity. In the presence of insulin, the binding of Chfr to Grb14 activates its ligase activity, leading to Aurora A and Polo-like kinase degradation and blocking cell division. Collectively, our results show that Chfr and Grb14 collaborate in a negative feedback loop controlling insulin-stimulated cell division., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Unlabelled: β-catenin signaling can be both a physiological and oncogenic pathway in the liver. It controls compartmentalized gene expression, allowing the liver to ensure its essential metabolic function. It is activated by mutations in 20%-40% of hepatocellular carcinomas (HCCs) with specific metabolic features. We decipher the molecular determinants of β-catenin-dependent zonal transcription using mice with β-catenin-activated or -inactivated hepatocytes, characterizing in vivo their chromatin occupancy by T-cell factor (Tcf)-4 and β-catenin, transcriptome, and metabolome. We find that Tcf-4 DNA bindings depend on β-catenin. Tcf-4/β-catenin binds Wnt-responsive elements preferentially around β-catenin-induced genes. In contrast, genes repressed by β-catenin bind Tcf-4 on hepatocyte nuclear factor 4 (Hnf-4)-responsive elements. β-Catenin, Tcf-4, and Hnf-4α interact, dictating β-catenin transcription, which is antagonistic to that elicited by Hnf-4α. Finally, we find the drug/bile metabolism pathway to be the one most heavily targeted by β-catenin, partly through xenobiotic nuclear receptors., Conclusions: β-catenin patterns the zonal liver together with Tcf-4, Hnf-4α, and xenobiotic nuclear receptors. This network represses lipid metabolism and exacerbates glutamine, drug, and bile metabolism, mirroring HCCs with β-catenin mutational activation., (© 2014 by the American Association for the Study of Liver Diseases.)

The action of insulin on metabolism and cell growth is mediated by a specific receptor tyrosine kinase, which, through phosphorylation of several substrates, triggers the activation of two major signaling pathways, the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the Ras/extracellular signal-regulated kinase (ERK) pathway. Insulin-induced activation of the receptor and downstream signaling is also subjected to a negative feedback control involving several mechanisms, among which the interaction of the insulin receptor and its substrates with inhibitory proteins. After summarizing the major mechanisms underlying the activation and attenuation of insulin signaling, this review focuses on its control by the Grb14 adaptor protein. Grb14 has been identif-ied as an inhibitor of insulin signaling and action, and is involved in insulin resistance associated with type 2 diabetes and obesity. Studies on the molecular mechanism of action of Grb14 have shown that, through interaction with the activated insulin receptor, Grb14 inhibits its catalytic activity and the activation of downstream signaling. However, the consequences of Grb14 gene invalidation are complex and tissue-specific, and some effects of Grb14 on insulin signaling appear to be linked to its interaction with effector proteins downstream the insulin receptor. Pharmacological inhibition of Grb14 should allow to enhance insulin sensitivity and improve energy homeostasis in insulin-resistant states., (© Société de Biologie, 2014.)

Epidemiological studies provide evidence for a close relationship between diabetes and cancer. Insulin is in fact a growth factor, and its binding to its membrane receptor activates intracellular signaling pathways involved in the regulation of both metabolism and cell proliferation. The balance between mitogenic and metabolic actions of insulin can be modulated by various mechanisms, including the way the ligand binds to its receptor or to the closely related insulin-like growth factor-1 (IGF-1) receptor. Cross-talks with other signaling pathways implicated in cell proliferation have also been described, like the Wnt/β catenin pathway, and involve the activation of common downstream effectors such as insulin receptor substrate-1 (IRS-1). Finally, the identification of new proteins activated by insulin and involved in intracellular signaling would allow a better understanding of the complex connections linking metabolic and proliferative regulatory pathways. As an example, the molecular adaptor Grb14, which is a specific inhibitor of insulin receptor catalytic activity, also controls insulin-induced metabolic and mitogenic signaling pathways through post-receptor mechanisms that remain to be fully elucidated., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) is an emerging epidemic disease. It represents a large spectrum of liver diseases, and affects both adults and children. The etiology of NAFLD is multifactorial. Indeed, several events such as caloric imbalance including sedentary lifestyle, obesity and/or a predisposing genetic background are key players in the increasing risk for NAFLD development and its progression. Recently, a sequence variation within the gene encoding for patatin-like phospholipase containing 3 (PNPLA3, rs738409) was found to modulate steatosis, inflammation and fibrosis in NAFLD. It was also demonstrated as a novel genetic marker associated with progressive ALD (alcoholic liver disease). In this mini-review, we summarize the current knowledge on (i) PNPLA3 variant(s) in the pathogenesis of liver diseases, and (ii) PNPLA3 gene regulation and potential function in liver., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

The effects of insulin and type 1 insulin-like growth factor (IGF-1) on metabolism, growth and survival are mediated by their association with specific receptor tyrosine kinases, which results in both receptor and substrate phosphorylation. Phosphotyrosine residues on receptors and substrates provide docking sites for signaling proteins containing SH2 (Src homology 2) domains, including molecular adaptors. This review focuses on the regulation of insulin/IGF-1 signaling and action by two adaptor families with a similar domain organization: the growth factor receptor-bound proteins Grb7/10/14 and the SH2B proteins. Both Grb10/14 and SH2B1/B2 associate with the activation loop of insulin/IGF-1 receptors through their SH2 domains, but association of Grb10/14 also involves their unique BPS domain. Consistent with Grb14 binding as a pseudosubstrate to the kinase active site, insulin/IGF-induced activation of receptors and downstream signaling pathways in cultured cells is inhibited by Grb10/14 adaptors, but is potentiated by SH2B1/B2 adaptors. Accordingly, Grb10 and Grb14 knockout mice show improved insulin/IGF sensitivity in vivo, and, for Grb10, overgrowth and increased skeketal muscle and pancreatic β-cell mass. Conversely, SH2B1-depleted mice display insulin and IGF-1 resistance, with peripheral depletion leading to reduced adiposity and neuronal depletion leading to obesity through associated leptin resistance. Grb10/14 and SH2B1 adaptors also modulate insulin/IGF-1 action by interacting with signaling components downstream of receptors and exert several tissue-specific effects. The identification of Grb10/14 and SH2B1 as physiological regulators of insulin signaling and action, together with observations that variants at their gene loci are associated with obesity and/or insulin resistance, highlight them as potential therapeutic targets for these conditions., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Background & Aims: The adiponutrin/PNPLA3 (patatin-like phospholipase domain-containing protein 3) variant I148M has recently emerged as an important marker of human fatty liver disease. In order to understand the role of the adiponutrin/PNPLA3 protein, we investigated the regulation of its expression in both human and mouse hepatocytes., Methods: Adiponutrin/PNPLA3 and lipogenic enzyme expression was determined by real-time PCR analysis in a wide panel of analysis in vivo in the mouse liver and in vitro in murine hepatocytes and human hepatocyte cell lines infected with ChREBP or SREBP1c-expressing adenoviruses., Results: We show that in the mouse liver, adiponutrin/PNPLA3 gene expression is under the direct transcriptional control of ChREBP (carbohydrate-response element-binding protein) and SREBP1c (sterol regulatory element binding protein1c) in response to glucose and insulin, respectively. In silico analysis revealed the presence of a ChoRE (carbohydrate response element) and of a SRE (sterol response element) binding site on the mouse adiponutrin/PNPLA3 gene promoter. Point mutation analysis in reporter gene assays identified the functional response of these two binding sites in the mouse adiponutrin/PNPLA3 promoter. In contrast, in human immortalized hepatocytes and in HepG2 hepatoma cells, only SREBP1c was able to induce adiponutrin/PNPLA3 expression, whereas ChREBP was unable to modulate its expression., Conclusions: All together, our results suggest that adiponutrin/PNPLA3 is regulated by two key factors of the glycolytic and lipogenic pathways, raising the question of its implication in the metabolism of carbohydrates and lipids., (Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Objective: Carbohydrate-responsive element-binding protein (ChREBP) is a key transcription factor that mediates the effects of glucose on glycolytic and lipogenic genes in the liver. We have previously reported that liver-specific inhibition of ChREBP prevents hepatic steatosis in ob/ob mice by specifically decreasing lipogenic rates in vivo. To better understand the regulation of ChREBP activity in the liver, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAc or O-GlcNAcylation), an important glucose-dependent posttranslational modification playing multiple roles in transcription, protein stabilization, nuclear localization, and signal transduction., Research Design and Methods: O-GlcNAcylation is highly dynamic through the action of two enzymes: the O-GlcNAc transferase (OGT), which transfers the monosaccharide to serine/threonine residues on a target protein, and the O-GlcNAcase (OGA), which hydrolyses the sugar. To modulate ChREBP(OG) in vitro and in vivo, the OGT and OGA enzymes were overexpressed or inhibited via adenoviral approaches in mouse hepatocytes and in the liver of C57BL/6J or obese db/db mice., Results: Our study shows that ChREBP interacts with OGT and is subjected to O-GlcNAcylation in liver cells. O-GlcNAcylation stabilizes the ChREBP protein and increases its transcriptional activity toward its target glycolytic (L-PK) and lipogenic genes (ACC, FAS, and SCD1) when combined with an active glucose flux in vivo. Indeed, OGT overexpression significantly increased ChREBP(OG) in liver nuclear extracts from fed C57BL/6J mice, leading in turn to enhanced lipogenic gene expression and to excessive hepatic triglyceride deposition. In the livers of hyperglycemic obese db/db mice, ChREBP(OG) levels were elevated compared with controls. Interestingly, reducing ChREBP(OG) levels via OGA overexpression decreased lipogenic protein content (ACC, FAS), prevented hepatic steatosis, and improved the lipidic profile of OGA-treated db/db mice., Conclusions: Taken together, our results reveal that O-GlcNAcylation represents an important novel regulation of ChREBP activity in the liver under both physiological and pathophysiological conditions.

To decipher the mechanism involved in Grb14 binding to the activated fibroblast growth factor receptor (FGFR), we used the bioluminescence resonance energy transfer (BRET) technique and the Xenopus oocyte model. We showed that Grb14 was recruited to FGFR1 into a trimeric complex containing also phospholipase C gamma (PLCγ). The presence of Grb14 altered FGF-induced PLCγ phosphorylation and activation. Grb14-FGFR interaction involved the Grb14-SH2 domain and the FGFR pY766 residue, which is the PLCγ binding site. Our data led to a molecular model whereby Grb14 binding to the phosphorylated FGFR induces a conformational change that unmasks a PLCγ binding motif on Grb14, allowing trapping and inactivation of PLCγ., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Background/aim: Estrogen-independent breast cancer cell growth is under the control of fibroblast growth factors receptors (FGFRs), but the role of phospholipase C gamma (PLC(gamma)) and Akt, the downstream effectors activated by FGFRs, in cell proliferation is still unresolved., Materials and Methods: FGFRs from highly invasive MDA-MB-231 cells were expressed in Xenopus oocyte, a powerful model system to assess the G(2)/M checkpoint regulation. Under FGF1 stimulation, an analysis of the progression in the M-phase of the cell cycle and of the Akt signaling cascades were performed using the phosphatidylinositol-3-kinase inhibitor, LY294002, and a mimetic peptide of the SH3 domain of PLC(gamma)., Results: Activated Akt binds and phosphorylates PLC(gamma) before Akt targets the tumor suppressor Chfr. Disruption of the Akt-PLC(gamma) interaction directs Akt binding to Chfr and accelerates the alleviation of the G(2)/M checkpoint., Conclusion: The PLC(gamma)-Akt interaction, triggered by FGF receptors from estrogen-independent breast cancer cells MDA-MB-231, regulates progression in the M-phase of the cell cycle.

Grb14 belongs to the Grb7 family of molecular adapters and was identified as an inhibitor of insulin signaling. Grb14 binds to activated insulin receptors (IR) and inhibits their catalytic activity. To gain more insight into the Grb14 molecular mechanism of action, we generated various mutants and studied the Grb14-IR interaction using coimmunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments. Biological activity was further analyzed using the Xenopus oocyte model and a functional complementation assay measuring cellular proliferation rate in Grb14 knockout mouse embryonic fibroblasts. These studies identified two important interaction sites, Grb14 L404-IR L1038 and Grb14 R385-IR K1168, involving the IR alphaC-helix and activation loop, respectively. Interestingly, the former involves residues that are likely to be crucial for the specificity of IR binding with regard to other members of the Grb7 family. In addition, mutation of the Grb14-S370 residue suggested that its phosphorylation status controlled the biological activity of the protein. We further demonstrated that insulin-induced Grb14-PDK1 interaction is required in addition to Grb14-IR binding to mediate maximal inhibition of insulin signaling. This study provides important insights into the molecular determinants of Grb14 action by demonstrating that Grb14 regulates insulin action at two levels, through IR binding and by interfering with downstream pathways. Indeed, a precise knowledge of the molecular mechanism of insulin signaling inhibition by Grb14 is a prerequisite for the development of insulin-sensitizing molecules to treat pathophysiological states such as obesity or type 2 diabetes.

The molecular adaptor Grb14 binds in vitro to the activated insulin receptor (IR) and inhibits IR signaling. In this study, we have used rat liver subcellular fractionation to analyze in vivo insulin effects on Grb14 compartmentalization and IR phosphorylation and activity. In control rats, Grb14 was recovered mainly in microsomal and cytosolic fractions, but was also detectable at low levels in plasma membrane and Golgi/endosome fractions. Insulin injection led to a rapid and dose-dependent increase in Grb14 content, first in the plasma membrane fraction, and then in the Golgi/endosome fraction, which paralleled the increase in IR beta-subunit tyrosine phosphorylation. Upon sustained in vivo IR tyrosine phosphorylation induced by high-affinity insulin analogs, in vitro IR dephosphorylation by endogenous phosphatases, and in vivo phosphorylation of the IR induced by injection of bisperoxo(1,10 phenanthroline)oxovanadate, a phosphotyrosine phosphatase inhibitor, we observed a striking correlation between IR phosphorylation state and Grb14 content in both the plasma membrane and Golgi/endosome fractions. In addition, coimmunoprecipitation experiments provided evidence that Grb14 was associated with phosphorylated IR beta-subunit in these fractions. Altogether, these data support a model whereby insulin stimulates the recruitment of endogenous Grb14 to the activated IR at the plasma membrane, and induces internalization of the Grb14-IR complex in endosomes. Removal of Grb14 from fractions of insulin-treated rats by KCl treatment led to an increase of in vivo insulin-stimulated IR tyrosine kinase activity, indicating that endogenous Grb14 exerts a negative feedback control on IR catalytic activity. This study thus demonstrates that Grb14 is a physiological regulator of liver insulin signaling.

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.

The peroxisome proliferator-activated receptor alpha (PPARalpha) belongs to the nuclear receptor family and plays a central role in the regulation of lipid metabolism, glucose homeostasis and inflammatory processes. In addition to its ligand-induced activation, PPARalpha is regulated by phosphorylation via ERK-MAPK, PKA and PKC. In this study we examined the effect of p38-MAPK on PPARalpha transcriptional activity. In COS-7 cells, anisomycin, a p38 activator, induced a dose-dependent phosphorylation of PPARalpha and a 50% inhibition of its transcriptional activity. In H4IIE hepatoma cells, anisomycin-induced p38 phosphorylation decreased both endogenous and PPARalpha ligand-enhanced L-CPTI and ACO gene expression. Interestingly, PPARalpha/p38 interaction required the molecular adapter ZIP/p62. Reducing ZIP/p62 expression by siRNA, partially reversed the inhibitory effect of anisomycin on L-CPTI gene expression. In conclusion, we showed that p38 activation induced PPARalpha phosphorylation and inhibition of its transcriptional activity through a trimeric interaction between p38-MAPK, ZIP/p62 and PPARalpha.

Adaptor proteins Grb7 and Grb2 have been implicated as being 2 potential therapeutic targets in several human cancers, especially those that overexpress ErbB2. These 2 proteins contain both a SH2 domain (Src homology 2) that binds to phosphorylated tyrosine residues contained within ErbB2 and other specific protein targets. Two assays based on enzyme-linked immunosorbent assay and fluorescence polarization methods have been developed and validated to find and rank inhibitors for both proteins binding to the pY(1139). Fluorescence polarization assays allowed the authors to determine quickly and reproducibly affinities of peptides from low nanomolar to high micromolar range and to compare them directly for Grb7 and Grb2. As a result, the assays have identified a known peptidomimetic Grb2 SH2 inhibitor (mAZ-pTyr-(alphaMe)pTyr-Asn-NH(2)) that exhibits the most potent affinity for the Grb7 SH2 domain described to date.

Grb14 is a molecular adaptor that binds to the activated insulin receptor (IR) and negatively regulates insulin signaling. We have studied the dynamics of interaction of the IR with Grb14, in real time, in living HEK cells, using bioluminescence resonance energy transfer (BRET). Insulin rapidly and dose-dependently stimulated this interaction. Removing insulin from the incubation medium only resulted in a modest decrease in BRET signal, indicating that the interaction between the IR and Grb14 can remain long after insulin stimulus has disappeared. BRET saturation experiments indicated that insulin markedly increases the affinity between IR and Grb14, resulting in recruitment of the adaptor to the activated IR. In addition, using both BRET and co-immunoprecipitation experiments, we demonstrated that insulin induced the dimerization of Grb14, most likely as a result of simultaneous binding of two Grb14 molecules on the activated IR. We also investigated the relationships between IR, Grb14 and the protein tyrosine phosphatase PTP1B. We observed that insulin-induced BRET between the IR and PTP1B was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the insulin receptor, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the ERK pathway. Our work suggests that Grb14 may regulate signalling through the insulin receptor by controlling its tyrosine-dephosphorylation in a site-specific manner.

The dynamics of interaction of the insulin receptor (IR) with Grb14 was monitored, in real time, in living human embryonic kidney cells, using bioluminescence resonance energy transfer (BRET). We observed that insulin rapidly and dose-dependently stimulated this interaction. We also observed that insulin-induced BRET between the IR and protein tyrosine phosphatase 1B (PTP1B) was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the IR, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the extracellular signal-regulated kinase pathway. Increased Grb14 expression in human liver-derived HuH7 cells also seemed to specifically decrease the phosphorylation of Y972. Our work therefore suggests that Grb14 may regulate signalling through the IR by controlling its tyrosine dephosphorylation in a site-specific manner.

Background: Fibroblast growth factors receptors (FGFRs) are involved in estrogen-independent breast cancer cell growth. Grbl4, a member of the Grb7 family of adapters, is an inhibitor of FGFR signaling., Materials and Methods: FGFR from highly invasive MDA-MB-231 cells were expressed in Xenopus oocyte, a widely used model system to question cascade transduction regulations. The effect of microinjection of Grb14 and various mimetic peptides for FGFR tyrosine residues were analysed by FGFR immunoprecipitation and Western blot analysis of signaling cascades., Results: PLCy, ERK2, JNK1 and AKT were blocked by Grb14. Only the pY766 phosphopeptide mimetic of the PLCgamma binding site on FGFR released the inhibitory action of Grb14., Conclusion: Grb14 binds to the Y766 site of MDA-MB-231-FGFR, competing for PLCy activation, thus inducing an arrest of the signaling transduction cascades.

Liver carnitine palmitoyl transferase (L-CPT) I is a key regulatory enzyme of long-chain fatty acid (LCFA) oxidation that ensures the first step of LCFA import into the mitochondrial matrix. In rat hepatocytes, we showed previously that L-CPT I gene expression was induced by LCFAs as well as by fibrates. The aim of this study was to determine whether LCFA-induced L-CPT I gene expression was mediated by PPARalpha. For this purpose, we constructed a PPARalpha-dominant negative receptor to inhibit endogenous PPARalpha signaling. Highly conserved hydrophobic and charged residues (Leu459 and Glu462) in helix 12 of the ligand-binding domain were mutated to alanine. These mutations led to a total loss of transcriptional activity due to impaired coactivator recruitment. Furthermore, competition studies confirmed that the mutated PPARalpha receptor abolished the wild-type PPARalpha receptor action and thus acted as a powerful dominant negative receptor. When overexpressed in rat hepatoma cells (H4IIE) using a recombinant adenovirus, the mutated PPARalpha receptor antagonized the clofibrate-induced L-CPT I gene expression, whereas it did not affect LCFA-induced L-CPT I. These results provide the first direct demonstration that LCFAs regulate L-CPT I transcription through a PPARalpha-independent pathway, at least in hepatoma cells.

Grb14 is an effector of insulin signaling, which directly inhibits insulin receptor catalytic activity in vitro. Here, we investigated whether the expression of Grb14 and its binding partner ZIP (PKC zeta interacting protein) is regulated during insulin resistance in type 2 diabetic rodents and humans. Grb14 expression was increased in adipose tissue of both ob/ob mice and Goto-Kakizaki (GK) rats, whereas there was no difference in liver. An increase was also observed in subcutaneous adipose tissue of type 2 diabetic subjects when compared with controls. ZIP expression was increased in adipose tissue of ob/ob mice and type 2 diabetic patients, but it did not vary in GK rats. Hormonal regulation of Grb14 and ZIP expression was then investigated in 3T3-F442A adipocytes. In this model, insulin stimulated Grb14 expression, while TNF-alpha increased ZIP expression. Moreover, the insulin-sensitizing drugs thiazolidinediones (TZDs) decreased Grb14 expression in 3T3-F442A adipocytes. Finally, we investigated the dynamic regulation of Grb14 expression in ob/ob mice in several conditions improving their insulin sensitivity. Prolonged fasting and treatment with metformin significantly decreased Grb14 expression in peri-epidydimal adipose tissue, while there was only a trend to a diminution after TZD treatment. Taken together, these results suggest that the regulation of Grb14 expression in adipose tissue may play a physiological role in insulin sensitivity.

Grb14 is the last described member of the Grb7 family of adaptors, containing Grb7, Grb10 and Grb14. These proteins share a series of conserved domains involved in protein-protein and protein-lipid interactions: an amino terminal proline-rich region, a C-terminal SH2 domain, and a central GM region containing a RA, a PH domain, and a newly described PIR (BPS) region. As shown for the other members of the Grb7/10/14 family, Grb14 binds to various receptor tyrosine kinases (RTKs) under ligand induction. This interaction involves the SH2 and PIR domains, and the respective participation of these domains is likely to be a determinant in the specificity of action of Grb14. At the present time, a role for this Grb14-RTK interaction was established only for insulin (IR) and FGF receptors (FGFR). Grb14, through its PIR, is an inhibitor of IR tyrosine kinase activity and thus of insulin effects. Grb14 also decreases FGF signaling, but more probably by interfering with cellular effectors downstream from the receptor. Only a few cytosolic partners of Grb14 are identified. One of them, the adaptor ZIP, allows phosphorylation of Grb14, and regulation of its inhibitory action on IR signaling. The identification of further proteins interacting with Grb14 is required to elucidate the biological role of this protein.

White adipose tissue (WAT) plays a critical role in the development of insulin resistance via secretion of free fatty acids (FFA) and adipocytokines. Muscle-specific insulin receptor knockout (MIRKO) mice do not develop insulin resistance or diabetes under physiological conditions despite a marked increase in adiposity and plasma FFA. On the contrary, WAT of MIRKO is sensitized to insulin action during a euglycemic clamp, and WAT glucose utilization is dramatically increased. To get insight into the potential antidiabetic role of MIRKO adiposity, we have studied insulin action in WAT during a euglycemic, hyperinsulinemic clamp, and we have characterized the morphology and biology of WAT. During the clamp, there is no alteration in the expression or activation in the insulin signaling molecules involved in glucose transport through the phosphoinositide 3-kinase/Akt and CAP/Cbl pathways in WAT from MIRKO. The 53% increase in WAT mass results from a 48% increase in adipocyte number (P < 0.05) without alteration in cell size and contemporary to a 300% increase in mRNA levels of the adipogenic transcription factor CCAAT enhancer binding protein-alpha (C/EBP-alpha) (P < 0.05). There is a 39.5% increase in serum adiponectin (P < 0.01) without modification in serum leptin, resistin, and TNF-alpha. In conclusion, the MIRKO mouse displays muscle insulin resistance, visceral obesity, and dyslipidemia but does not develop hyperinsulinemia or diabetes. There is an accelerated differentiation of small insulin sensitive adipocytes, an increased secretion of the insulin sensitizer adiponectin, and maintenance of leptin sensitivity. The MIRKO mouse confirms the importance of WAT plasticity in the maintenance of whole body insulin sensitivity and represents an interesting model to search for new secreted molecules that positively alter adipose tissue biology.

Grb14 belongs to the Grb7 family of adapter proteins and was identified as a negative regulator of insulin signal transduction. Its inhibitory effect on the insulin receptor kinase activity is controlled by a newly discovered domain called PIR. To investigate the biochemical and biophysical characteristics of this new domain, we cloned and purified recombinant PIR-SH2, PIR, and SH2 domains. The isolated PIR and PIR-SH2 domains were physiologically active and inhibited insulin-induced reinitiation of meiosis in the Xenopus oocytes system. However, NMR experiments on (15)N-labelled PIR revealed that it did not present secondary structure. These results suggest that the PIR domain belongs to the growing family of intrinsically unstructured proteins.

The role of Grb7 adapters, Grb7, Grb10, and Grb14, was investigated in Xenopus oocytes expressing fibroblast growth factor receptors (FGFR). FGF-induced maturation of FGFR-expressing oocytes was blocked by previous injection of Grb7 or Grb14, but not Grb10. This effect correlated with Grb7/14 binding to the receptor, and inhibition of the Ras-dependent pathway. Interestingly, the phosphorylated insulin receptor interacting region (PIR) and Src 2 homology domains (SH2) of Grb7 and Grb14 were differently implicated in the inhibition of FGFR signalling. This study provided further evidence for specificity of the biological action of the Grb7 adapters on receptor tyrosine kinase signalling.

Grb14 is a member of the Grb7 family of adapters and acts as a negative regulator of insulin-mediated signaling. Here we found that the protein kinase Czeta (PKCzeta) interacting protein, ZIP, interacted with Grb14. Coimmunoprecipitation experiments demonstrated that ZIP bound to both Grb14 and PKCzeta, thereby acting as a link in the assembly of a PKCzeta-ZIP-Grb14 heterotrimeric complex. Mapping studies indicated that ZIP interacted through its ZZ zinc finger domain with the phosphorylated insulin receptor interacting region (PIR) of Grb14. PKCzeta phosphorylated Grb14 under in vitro conditions and in CHO-IR cells as demonstrated by in vivo labeling experiments. Furthermore, Grb14 phosphorylation was increased under insulin stimulation, suggesting that the PKCzeta-ZIP-Grb14 complex is involved in insulin signaling. The PIR of Grb14, which also interacts with the catalytic domain of the insulin receptor (IR) and inhibits its activity, was preferentially phosphorylated by PKCzeta. Interestingly, the phosphorylation of Grb14 by PKCzeta increased its inhibitory effect on IR tyrosine kinase activity in vitro. The role of ZIP and Grb14 in insulin signaling was further investigated in vivo in Xenopus laevis oocytes. In this model, ZIP potentiated the inhibitory action of Grb14 on insulin-induced oocyte maturation. Importantly, this effect required the recruitment of PKCzeta and the phosphorylation of Grb14, providing in vivo evidences for a regulation of Grb14-inhibitory action by ZIP and PKCzeta. Together, these results suggest that Grb14, ZIP, and PKCzeta participate in a new feedback pathway of insulin signaling.

Glucose is required for an efficient expression of the glucose transporter GLUT2 and other genes. We have shown previously that the intracytoplasmic loop of GLUT2 can divert a signal, resulting in the stimulation of glucose-sensitive gene transcription. In the present study, by interaction with the GLUT2 loop, we have cloned the rat karyopherin alpha2, a receptor involved in nuclear import. The specificity of the binding was restricted to GLUT2, and not GLUT1 or GLUT4, and to karyopherin alpha2, not alpha1. When rendered irreversible by a cross-linking agent, this transitory interaction was detected in vivo in hepatocytes. A role for karyopherin alpha2 in the transcription of two glucose-sensitive genes was investigated by transfection of native and inactive green fluorescent protein-karyopherin alpha2 in GLUT2-expressing hepatoma cells. The amount of inactive karyopherin alpha2 receptor reduced, in a dose-dependent manner, the GLUT2 and liver pyruvate kinase mRNA levels by competition with endogenous active receptor. In contrast, the overexpression of karyopherin alpha2 did not significantly stimulate GLUT2 and liver pyruvate kinase mRNA accumulation in green fluorescent protein-sorted cells. The present study suggests that, in concert with glucose metabolism, karyopherin alpha2 transmits a signal to the nucleus to regulate glucose-sensitive gene expression. The transitory tethering of karyopherin alpha2 to GLUT2 at the plasma membrane might indicate that the receptor can load the cargo to be imported locally.

Grb14 belongs to the Grb7 family of adapters and was recently identified as a partner of the insulin receptor (IR). Here we show that Grb14 inhibits in vitro IR substrate phosphorylation. Grb14 does not alter the K(m) for ATP and behaves as an uncompetitive inhibitor for the IR substrate. Similar experiments performed with other members of the Grb7 family, Grb7 and Grb10, and with IGF-1 receptor argue in favor of a specific inhibition of the IR catalytic activity by Grb14. The IR-interacting domain of Grb14, the PIR, is sufficient for the inhibitory effect of Grb14, whereas the SH2 domain has no effect on IR catalytic activity. In Chinese hamster ovary (CHO) cells overexpressing both IR and Grb14, Grb14 binds to the IR as early as 1 min after insulin stimulation, and the two proteins remain associated. When interacting with Grb14, the IR is protected against tyrosine phosphatases action and therefore maintained under a phosphorylated state. However, the binding of Grb14 to the IR induces an early delay in the activation of Akt and ERK1/2 in CHO-IR cells, and ERK1/2 are less efficiently phosphorylated. These findings show that Grb14 is a direct inhibitor of the IR catalytic activity and could be considered as a modulator of insulin signaling.

A novel protein was cloned from a rat liver cDNA library by interaction with the liver glucokinase. This protein contained 339 residues and possessed a canonical consensus sequence for a dual specificity phosphatase. The recombinant protein was able to dephosphorylate phosphotyrosyl and phosphoseryl/threonyl substrates. We called this protein the glucokinase-associated phosphatase (GKAP). The GKAP partially dephosphorylated the recombinant glucokinase previously phosphorylated, in vitro, by protein kinase A. The GKAP fused with green fluorescent protein was located in the cytosol, where glucokinase phosphorylates glucose, and not in the nucleus where the glucokinase is retained inactive by the glucokinase regulatory protein. More importantly, the GKAP accelerated the glucokinase activity in a dose-dependent manner and with a stoichiometry compatible with a physiological mechanism. This strongly suggested that the interaction between GKAP and glucokinase had a functional significance. The cloning of this novel protein with a dual specificity phosphatase activity allows the description of a possible new regulatory step in controlling the glycolysis flux.

The molecular adapter Grb7 is likely to be implicated in the development of certain cancer types. In this study we show that Grb7 binds the insulin receptors, when they are activated and tyrosine phosphorylated. This interaction is documented by two-hybrid experiments, GST pull-down assays and in vivo coimmunoprecipitations. In addition, our results argue in favor of a preferential association between Grb7 and the insulin receptors when compared to other tyrosine kinase receptors like the EGF receptor, the FGF receptor and Ret. Interestingly, Grb7 is not a substrate of the insulin receptor tyrosine kinase activity. Grb7 binds the activated tyrosine kinase loop of the insulin receptors. Two domains of Grb7 are implicated in the insulin receptor binding: the SH2 domain and the PIR (phosphotyrosine interacting region). The role of these two domains in the interaction with the insulin receptor was already reported for Grb10 and Grb14, the other members of the Grb7 family of proteins. However, the relative importance of these domains varies, considering the receptor and the Grb protein. These differences should be a determinant of the specificity of the receptor tyrosine kinase-Grbs binding, and thus of the implication of Grb7/10/14 in signal transduction.