Anne-Françoise Burnol

Glucose Utilization Rates and Insulin Sensitivity In Vivo in Tissues of Virgin and Pregnant Rats

In vivo studies have shown that insulin resistance in late pregnancy results from a decreased sensitivity of liver and peripheral tissues. In the present study, measurements of the rates of glucose utilization by skeletal muscles (soleus, extensor digitorum longus, epitrochlearis, and diaphragm), white adipose tissue, and brain of virgin and 19-day pregnant rats were performed in the basal condition and during a euglycemic, hyperinsulinemic (400 μU/ml) clamp to quantify the partition of glucose utilization and to identify the tissues other than liver responsible for insulin resistance. Fetal and placental glucose utilization rates were also measured in pregnant rats. The fetal glucose utilization rate (22 mg/min/kg) was very high and was not stimulated by physiologic maternal hyperinsulinemia. By contrast, the placental glucose utilization rate (29 mg/ min/kg) was increased by 30% during hyperinsulinemia. The glucose utilization rate of the conceptus represented 23% of the maternal glucose utilization rate in the basal state. Glucose utilization rates in the basal condition were not statistically altered by pregnancy in brain, skeletal muscles, and white adipose tissue. During hyperinsulinemia (400 μU/ml), glucose utilization rates in extensor digitorum longus, epitrochlearis, and white adipose tissue were 30–70% lower in pregnant than in virgin rats. Insulin sensitivity of glucose metabolism in all the tissues tested other than brain was 50% lower in pregnant than in virgin rats. We conclude that skeletal muscles and, to a smaller extent, adipose tissue are involved in the insulin resistance of late pregnancy.

O-GlcNAcylation Increases ChREBP Protein Content and Transcriptional Activity in the Liver

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 ChREBPOG 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 ChREBPOG 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, ChREBPOG levels were elevated compared with controls. Interestingly, reducing ChREBPOG 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.

Identification of the Rat Adapter Grb14 as an Inhibitor of Insulin Actions

We cloned by interaction with the β-subunit of the insulin receptor the rat variant of the human adapter Grb14 (rGrb14). rGrb14 is specifically expressed in rat insulin-sensitive tissues and in the brain. The binding of rGrb14 to insulin receptors is insulin-dependent in vivo in Chinese hamster ovary (CHO) cells overexpressing both proteins and importantly, in rat liver expressing physiological levels of proteins. However, rGrb14 is not a substrate of the tyrosine kinase of the receptor. In the two-hybrid system, two domains of rGrb14 can mediate the interaction with insulin receptors: the Src homology 2 (SH2) domain and a region between the PH and SH2 domains that we named PIR (forphosphorylated insulin receptor-interactingregion). In vitro interaction assays using deletion mutants of rGrb14 show that the PIR, but not the SH2 domain, is able to coprecipitate insulin receptors, suggesting that the PIR is the major binding domain of rGrb14. The interaction between rGrb14 and the insulin receptors is almost abolished by mutating tyrosine residue Tyr1150 or Tyr1151 of the receptor. The overexpression of rGrb14 in CHO-IR cells decreases insulin stimulation of both DNA and glycogen synthesis. These effects are accompanied by a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1, but insulin receptor autophosphorylation is unaltered. These findings suggest that rGrb14 could be a new downstream signaling component of the insulin-mediated pathways.

Inhibition of insulin receptor catalytic activity by the molecular adapter Grb14

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.

Distinct regulation of adiponutrin/PNPLA3 gene expression by the transcription factors ChREBP and SREBP1c in mouse and human hepatocytes

BACKGROUND & AIMSnThe 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.nnnMETHODSnAdiponutrin/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.nnnRESULTSnWe 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.nnnCONCLUSIONSnAll 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.

Increased adipose tissue expression of Grb14 in several models of insulin resistance

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‐α 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.

Evidence for an interaction between the insulin receptor and Grb7. A role for two of its binding domains, PIR and SH2

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.

A novel cytosolic dual specificity phosphatase, interacting with glucokinase, increases glucose phosphorylation rate.

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 Adapter Protein ZIP Binds Grb14 and Regulates Its Inhibitory Action on Insulin Signaling by Recruiting Protein Kinase Cζ

ABSTRACT 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 Cζ (PKCζ) interacting protein, ZIP, interacted with Grb14. Coimmunoprecipitation experiments demonstrated that ZIP bound to both Grb14 and PKCζ, thereby acting as a link in the assembly of a PKCζ-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. PKCζ 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 PKCζ-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 PKCζ. Interestingly, the phosphorylation of Grb14 by PKCζ 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 PKCζ and the phosphorylation of Grb14, providing in vivo evidences for a regulation of Grb14-inhibitory action by ZIP and PKCζ. Together, these results suggest that Grb14, ZIP, and PKCζ participate in a new feedback pathway of insulin signaling.

In Vivo Glucose Utilization in Rat Tissues During the Three Phases of Starvation

Three phases of starvation have been described from changes in protein and lipid utilization in birds and mammals. In the present study, tissue glucose utilization was measured in vivo during these three phases, using a 2-deoxy-[1-3H]glucose technique in the anesthetized rat. According to this technique, the term glucose utilization therefore refers to transport and phosphorylation of glucose in tissues, ie, whatever is the fate of glucose. Whole-body glucose turnover rate, which was determined by a continuous infusion of [3-3H]glucose, decreased by 40% during the first two days of starvation (phase 1); it did not change thereafter, neither in the protein-sparing phase 2 nor in phase 3, which is marked by an increase in net protein breakdown. Two days of starvation caused a marked decrease in the glucose utilization in skeletal muscles; this decrease was higher in oxidative muscles (65% in diaphragm, 66% in soleus) than in glycolytic muscles (31% in extensor digitorum longus, 34% in epitrochlearis). Glucose utilization also decreased in heart atria (75%), heart ventricles (93%), and white adipose tissue (54%); by contrast, there was a two-fold increase in glucose utilization in brown adipose tissue and no change in brain and skin. No variations were observed in glucose utilization in any of the tissues from phase 1 to phase 2. However, phase 3 was marked by a decrease in glucose utilization in extensor digitorum longus (45%), brown adipose tissue (76%), brain (29%), and skin (40%), whereas there was a 2.3- and 3.4-fold increase in glucose utilization in diaphragm and heart ventricles, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)