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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 1, 2004 as doi:10.1096/fj.03-0824fje. |
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* Département d’Endocrinologie, Institut Cochin INSERM U 567-CNRS UMR 8104-Université René Descartes, Paris;
INSERM U 449 and Centre de Recherche en Nutrition Humaine de Lyon, Faculté de Médecine R. Laennec, Lyon;
MERCK-Santé, Centre de Recherche de Chilly-Mazarin; and
Service d’Endocrinologie, Diabétologie et Nutrition, Hôpital E. Herriot, Lyon, France
2Correspondence: Département d’Endocrinologie, Institut Cochin INSERM U 567-CNRS UMR 8104-Université René Descartes, 24 rue du Faubourg Saint-Jacques, 75674 Paris, France. E-mail: burnol{at}cochin.inserm.fr
SPECIFIC AIMS
Grb14 is a molecular adaptor expressed specifically in insulin-sensitive tissues which inhibits in vitro insulin receptor (IR) tyrosine kinase activity. Its binding partner, the atypical protein kinase C zeta (PKC
) interacting protein (ZIP), targets PKC activity toward Grb14, then increasing Grb14 inhibitory action on insulin signaling. We investigated whether regulation of Grb14 and ZIP expression is associated with insulin resistance and type 2 diabetes in vivo in rodents and humans.
PRINCIPAL FINDINGS
1. Grb14 and ZIP expression in insulin-sensitive tissues of ob/ob mice and Goto-Kakizaki (GK) rats
To determine the regulation of Grb14 and ZIP expression during insulin resistance in vivo, we used ob/ob mouse and GK rat as animal models of type 2 diabetes. Considering the relative Grb14 expression levels in the different insulin-sensitive tissues (Fig. 1
), we measured Grb14 and ZIP expression in liver and peri-epididymal adipose tissue of diabetic rodents and their appropriate controls by performing real-time quantitative RT-PCR and Western blot analysis. As shown in Fig. 1
, Grb14 mRNA expression was significantly increased in white adipose tissue of both ob/ob mice and GK rats by 106 ± 41% (P<0.05) and 112 ± 41% (P<0.05), respectively. Similarly, the expression of Grb14 protein in adipose tissue was increased by 72 ± 22% (P<0.05) in ob/ob mice and 75 ± 15% (P<0.01) in GK rats compared with controls. There was no difference in Grb14 mRNA or protein expression in liver of these rodents. Although ZIP expression did not vary in insulin-sensitive tissues of GK rats, it was increased in adipose tissue of ob/ob mice at mRNA and protein levels respectively by 69 ± 8% (P<0.01) and 65 ± 10% (P<0.05) compared with controls. There was no difference in ZIP expression in liver of ob/ob mice.
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2. Grb14 and ZIP mRNA expression in adipose tissue and muscle of type 2 diabetic patients
To further determine whether the results observed in rodents were valid in humans, we measured Grb14 and ZIP mRNA expression in subcutaneous adipose tissue of patients with type 2 diabetes. Since skeletal muscle is the main tissue contributing to glucose uptake in response to insulin after a glucose load in humans, we also checked the expression of these two genes in muscle samples of type 2 diabetic subjects. Basal Grb14 mRNA expression was significantly increased by 43 ± 10% (P<0.05) in adipose tissue of diabetic patients compared with healthy volunteers. Moreover, ZIP mRNA expression was 2.5-fold increased (P<0.01) in adipose tissue of these diabetic patients. Whereas Grb14 gene expression was not significantly altered in skeletal muscle of type 2 diabetic patients, ZIP mRNA concentration was increased by 50 ± 14% (P<0.01). These results show that basal Grb14 and ZIP expression levels are increased in adipose tissue of insulin-resistant states in humans as well as in rodents.
3. Regulation of Grb14 and ZIP expression in 3T3-F442A adipocytes
To investigate the regulation of Grb14 and ZIP gene expression in vitro, we incubated 3T3-F442A adipocytes with various factors known to affect insulin sensitivity. Treatment of these cells with 100 nM insulin for 24 h induced a 2.3-fold (P<0.01) increase in Grb14 mRNA expression whereas the ZIP mRNA level was decreased by 58 ± 2% (P<0.01) in the same conditions. A TNF
treatment (10 ng/mL for 24 h) significantly increased ZIP mRNA level by 95% ± 25% (P<0.01), but did not modify Grb14 mRNA expression. On the other hand, Grb14 and ZIP expression in adipocytes were not altered by a high concentration of glucose (25 mM) for 24 h. We examined the effect of insulin-sensitizing thiazolidinediones (TZD) on Grb14 and ZIP expression. Incubating 3T3-F442A adipocytes for 48 h with 1 µM pioglitazone decreased Grb14 mRNA and protein by respectively 67 ± 3% (P<0.01) and 64 ± 10% (P<0.01). The effect of pioglitazone was dose dependent, with a diminution of Grb14 expression at concentrations as low as 100 nM. Similarly, 1 µM troglitazone diminished the Grb14 protein level by 32 ± 8% (P<0.05). In contrast, TZD did not regulate ZIP mRNA expression in the same experimental conditions.
4. Dynamic regulation of Grb14 expression in vivo in ob/ob mice
To confirm the association between Grb14 level of expression and insulin sensitivity in vivo, we investigated the effect of prolonged fasting and insulin-sensitizing drugs such as TZD (rosiglitazone 5 mg·kg–1 ·day–1) and metformin (200 mg·kg–1·day–1) in ob/ob mice. These conditions normalized both plasma glucose and insulin concentrations. After 72 h of fasting or 2 days of metformin treatment, Grb14 expression in adipose tissue of ob/ob mice was significantly decreased by respectively 45 ± 8% (P=0.03) and 38 ± 16% (P=0.018). Conversely, there was only a tendency to a diminution of Grb14 in adipose tissue of rosiglitazone-treated ob/ob mice (P=0.16). Nevertheless, this tendency became statistically significant when we measured Grb14 expression in a visceral fat depot (perirenal adipose tissue), with a 35 ± 12% decrease in response to TZD treatment (P=0.04) (Fig. 2
). Rosiglitazone treatment also induced a 37% ± 8% (P<0.001) decrease in liver Grb14 expression. This effect appeared to be TZD specific since neither fasting nor metformin treatment altered Grb14 expression in liver.
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CONCLUSIONS AND SIGNIFICANCE
The prevalence of type 2 diabetes is expanding rapidly in most countries and is expected to affect 
6.5% of the world population in the next 20 years. A major hallmark of type 2 diabetes is resistance to insulin action in insulin-sensitive tissues. Alterations in the expression or action of intracellular effectors of insulin signaling have been shown to modulate peripheral tissue insulin sensitivity. There is growing evidence for a functional role of the Grb7 family of adapters in insulin signaling, especially Grb10 and Grb14. Although studies published on the effects of Grb10 overexpression in cultured cells provide conflicting results for stimulatory vs. inhibitory actions on insulin responses, it is well established that Grb14 decreased insulin receptor catalytic activity and subsequently insulin distal effects like DNA and glycogen synthesis. However, most of these data were obtained in vitro and in cellular models that were not metabolic targets of insulin. In the present study, we demonstrated that Grb14 gene and protein expression were increased in white adipose tissue of independent animal models of insulin resistance such as ob/ob mice and GK rats. Grb14 mRNA expression was also significantly increased in adipose tissue of type 2 diabetic subjects. The phenotype of Grb14-deficient mice recently reported strengthens the in vivo inhibitory action of Grb14 on insulin signaling.
The differential regulation of Grb14 expression in insulin target tissues is striking. Indeed, although Grb14 is expressed fivefold more in liver than in adipose tissue, we were unable to detect any difference in liver of diabetic rodents. However, Grb14 activity in liver can also be regulated at post-translational levels during insulin resistance. Supporting this, we demonstrated previously that Grb14 phosphorylation by PKC potentiated its inhibitory action in Xenopus oocyte. Subcellular localization of Grb14 may also be critical for its biological activity. Since muscle accounts for 60–70% of glucose disposal vs. only 10% for adipose tissue, this adipose tissue-selective regulation of Grb14 expression could moderate the physiological importance of Grb14 in insulin signaling. Nevertheless, growing data support the hypothesis that insulin resistance could be initiated in fat cells, leading secondarily to alterations in insulin action in muscle and liver. Since Grb14 is a potent inhibitor of insulin receptor catalytic activity in vitro, it is tempting to speculate that increased expression of Grb14 may participate in the development of insulin resistance (Fig. 3
). Further studies using Grb14 overexpression in adipose tissue are needed to assess the functional consequences on insulin signaling. Nevertheless, we demonstrated here that different conditions ameliorating ob/ob mice insulin sensitivity and hyperinsulinemia are accompanied by a substantial decrease in adipose tissue Grb14 expression. Furthermore, Grb14 expression in adipose tissue, as well as in adipocytes, is nicely negatively regulated by TZD, leading to the hypothesis that the anti-diabetic effect of TZD may be mediated at least in part through this effect. Thus, these data strongly support the hypothesis of a functional role of Grb14 in adipose tissue.
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Compared with Grb14, the modifications of ZIP expression in adipose tissue did not fully correlate with the variation in insulin sensitivity. Although ZIP expression was increased in type 2 diabetic subjects and ob/ob mice, there was no difference in GK rats. Moreover, TZD did not regulate ZIP expression. However, ZIP may interfere with insulin action by promoting the phosphorylation of Grb14 by PKC. Alternatively, ZIP may target a specific pool of PKC to other signaling molecules involved in insulin action, such as insulin receptor substrate-1 (IRS-1), or interfere with other signaling pathways, like TNF signaling, which alters insulin sensitivity.
In summary, the present study clearly demonstrates a link between the expression of Grb14 in adipose tissue and insulin sensitivity in vivo both in rodents and type 2 diabetic patients, supporting the hypothesis of a physiological role of Grb14 in insulin signaling. Although work is needed to confirm these results, Grb14 appears to be an interesting new therapeutic target for the treatment of insulin resistance in obesity and type 2 diabetes.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0824fje; 
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