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Previous Article | Next Article 
Molecular and Cellular Biology, September 2007, p. 6497-6505, Vol. 27, No. 18
0270-7306/07/$08.00+0 doi:10.1128/MCB.00679-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Departments of Pharmacology,1 Medicine,2 Biochemistry,3 Cellular and Structural Biology,5 Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229,6 Mammalian Genetics Section, GDDB, NIDDK, National Institutes of Health, Bethesda, Maryland 208924
Received 18 April 2007/ Returned for modification 20 June 2007/ Accepted 28 June 2007
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Insulin functions by binding to the extracellular 
-subunit of the insulin receptor (IR), which results in tyrosine phosphorylation and activation of the receptor tyrosine kinase in the ß-subunit. Activated IR tyrosine kinase phosphorylates cellular substrates, such as IR substrate 1/2 (IRS-1/2) and Shc, leading to the activation of the phosphatidlyinositol (PI) 3-kinase and mitogen-activated protein kinase (MAPK) signaling pathways, respectively. Tyrosine phosphorylation of IR also generates docking sites for cellular adaptor proteins that positively or negatively regulate insulin signaling to downstream targets to control glucose metabolism and energy homeostasis.
Grb10 is a pleckstrin homology (PH) and Src homology 2 (SH2) domain-containing adaptor protein that binds to tyrosine-phosphorylated IR in response to insulin stimulation (12, 14, 21). Grb10 belongs to the Grb7/10/14 family that also includes Grb7 (15) and Grb14 (7). In mice, the Grb10 gene is imprinted and mapped to chromosome 11. Maternal expression of Grb10 is observed in almost all organs except the brain, where it is biallelically expressed, with preferential paternal-allele expression in adulthood (14). Recent publications have demonstrated that overexpression and suppression of Grb10 expression result in opposite growth phenotypes (4, 23).
The role of Grb10 in insulin signaling remains controversial. In response to insulin stimulation, Grb10 binds to tyrosine-phosphorylated residues within the kinase domain of the IR via its SH2 and BPS (for between the PH and SH2 domains) domains (12, 14, 21). Numerous studies hsve shown that the binding of Grb10 to the kinase domain of the IR leads to reduced insulin signaling and action. For example, overexpression of Grb10 isoforms has been shown to inhibit insulin-stimulated activation of PI 3-kinase, Akt, and MAPK (9, 16, 26, 28). Overexpression of Grb10 has also been found to inhibit insulin downstream events, such as glycogen synthase activity (17) and glucose uptake (16). Consistent with a negative role for endogenous Grb10 in insulin/IGF-1 signaling, two recent studies showed that knocking down of Grb10 by small interfering RNA led to enhanced IGF-1 and insulin signaling (9, 13). However, there are a few studies that suggest that Grb10 is a positive regulator of the insulin-signaling pathway. It has been shown that overexpression of the SH2 domain of Grb10 inhibited insulin-stimulated downstream metabolic events in 3T3-L1 adipocytes and L6 cells (8), as well as mitogenesis in fibroblasts (19). These findings led to the conclusion that full-length Grb10 may play a positive role in insulin action. In support of this, overexpression of full-length mouse Grb10 has been reported to positively affect insulin-stimulated mitogenesis in fibroblasts (27).
The in vivo roles of Grb10 in insulin signaling and glucose homeostasis are largely unknown. Shiura et al. have demonstrated that overexpression of Grb10 in mice resulted in postnatal insulin resistance (23), consistent with the view that ablation of Grb10 may increase insulin sensitivity (12). Knocking out Grb10 in mice by the gene trap technique resulted in overgrowth of the embryo and the placenta, as well as an increase in the size of the animal at birth, suggesting an inhibitory role of endogenous Grb10 in embryonic growth and development (4).
In the present study, we report the generation of Grb10-deficent mice in which the imprinted Grb10 gene is disrupted in all tissues except the brain. We found that disruption of Grb10 gene expression led to postnatal overgrowth, consistent with a role for endogenous peripheral Grb10 as a growth suppressor. Disruption of Grb10 expression also led to increased insulin signaling in muscle and adipose tissues, as well as enhanced muscle insulin sensitivity. Taken together, our results provide strong evidence that peripheral Grb10 functions as a negative regulator of insulin signaling and action in vivo.
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MATERIALS AND METHODS |
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Glucose and insulin measurements.
The tail vein blood glucose level was measured by using an automatic glucometer (Rightest GM300; Bionime Corp). To measure the blood insulin level, tail vein blood was collected using heparinized capillary tubes (
75 µl). Blood samples were centrifuged, and the plasma insulin concentration was determined using an enzyme-linked immunosorbent assay (Mercodia, Sweden).
In vivo insulin action. Mice at 4 to 6 months of age were fasted overnight and intraperitoneally (i.p.) injected with insulin (1 U or 5 U/kg animal body weight; Humulin R; Eli Lilly, Indianapolis, IN) or an equal volume of saline. After 5 or 10 min, the mice were euthanized via cervical dislocation, and tissues were immediately excised, frozen in liquid nitrogen, and kept at –80°C until homogenization.
Tissue homogenization and Western blot analysis. Tissue samples were homogenized in ice-cold buffer containing 50 mM HEPES (pH 7.6), 150 mM sodium chloride, 20 mM sodium pyrophosphate, 20 mM beta-glycerophosphate, 10 mM sodium fluoride, 2 mM sodium orthovanadate, 2 mM EDTA, 1.0% Igepal (a nonionic, nondenaturing detergent), 10% glycerol, 2 mM phenylmethylsulfonyl fluoride, 1 mM magnesium chloride, 1 mM calcium chloride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Tissue homogenates were clarified by centrifugation, and protein concentrations in the supernatant were determined using the Bradford assay. Proteins in the supernatants of the tissue homogenates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Bound proteins were blocked with 1% nonfat dry milk in Tris-buffered saline-0.1% Tween 20 and probed with specific primary antibodies (a polyclonal antibody against Grb10 was raised in our laboratory; antibodies to Akt, MAPK, phospho-AktS473, and phospho-MAPK were obtained from Cell Signaling Technology; antibodies to Grb7 and Grb14 were purchased from Santa Cruz and Chemicon International, Inc., respectively). The membranes were then washed with Tris-buffered saline buffer with 0.5% Tween 20 and incubated with horseradish peroxidase- or alkaline phosphatase-conjugated second antibody (Promega Corp., WI). Proteins were visualized using chemiluminescence or alkaline phosphatase-conjugated reactions.
Body weight and body composition. Mouse body weight was measured on a weekly basis. To check body composition, mice were i.p. injected with Avertin (250 mg/kg animal body weight). Fat and lean tissue masses and bone mineral content were determined using dual-energy X-ray absorptiometry (DEXA) (GE Medical Systems and Lunar PIXImus 2, Madison WI).
Hyperinsulinemic-euglycemic clamp studies.
Hyperinsulinemic-euglycemic clamp studies were performed on animals fed with regular chow at 10 weeks of age (Teklad 7912; Harlan, Indianapolis, IN) (n = 5 to 6/group) or a high-fat diet (HFD) (45 % kcal from fat) (D12451'; Research Diets Inc., New Brunswick, NJ) for 10 weeks beginning at 10 weeks of age (n = 5 for wild-type and n = 3 for Grb10 knockout mice). For the clamp procedure, 3 to 5 days prior to the insulin clamp, mice were anesthetized using an i.p. injection of pentobarbital (60 mg/kg body weight), and a catheter was inserted into the heart (right atrium) of each mouse during a sterile surgical procedure as previously described (5). The mice were fasted for 5 h before the insulin clamp studies were performed. The insulin clamp procedure was done in awake, unrestrained, unstressed animals that had been implanted with a catheter. In order to determine the basal and insulin-stimulated rates of glucose appearance, a prime continuous infusion of [3-3H]glucose (10-µCi bolus; 0.1 µCi/min) was started at –60 min and was maintained throughout the 90-min insulin clamp study. At time zero, a primed continuous (18 mU/min·kg) infusion of human insulin was started simultaneously with a variable infusion of 10% dextrose in order to maintain the plasma glucose concentration constant at its basal level. Plasma samples (30 µl blood/sample) for determination of [3-3H]glucose specific activity and plasma insulin levels were obtained at times –20, –10, and 0 min during the basal period and at 30, 60, 70, 80, and 90 min during the insulin clamp period. To prevent anemia, red blood cells were resuspended in normal saline and then reinfused with an amount of saline to replace equivalently the amount of plasma withdrawn. Glucose concentrations were measured by the glucose oxidase method on a Beckman Glucose Analyzer II (Beckman, Fullerton, CA), and insulin levels were measured as described above. Data for total-body glucose uptake and hepatic glucose production (HGP) represent the mean values during the last 20 min of the basal period and during the last 30 min of the insulin clamp period. During the last 20 min of the basal period and during the last 30 min of the insulin clamp study, a steady-state plateau of [3-3H]glucose radioactivity was achieved in all studies. Under steady-state conditions, the rate of total-body glucose disappearance (Rd) equals the rate of total-body glucose appearance and was calculated by dividing the infusion rate of [3-3H]glucose (dpm) by the steady-state plateau of [3-3H]glucose specific activity (counts per milligram). The rate of HGP was calculated by subtracting the exogenous glucose infusion rate from the rate of total-body glucose appearance (18). Skeletal-muscle tissues were collected after the mice were euthanized.
Statistics. Quantification of the relative increase in insulin-stimulated protein phosphorylation was performed by analyzing Western blots using Scion Image software and was normalized with the amount of protein expression in each experiment. Statistical analysis of the data was performed using analysis of variance (ANOVA). Statistical significance was set at P values of <0.05, <0.01, and <0.001.
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RESULTS |
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To determine whether the disruption of the Grb10 gene is specific, we also investigated the expression of two other members of the Grb7/10/14 family, Grb7 and Grb14, in brain and peripheral insulin target tissues, such as fat, muscle, and liver. We found that Grb7 is predominantly expressed in the liver, and disruption of the Grb10 gene had no effect on Grb7 expression (Fig. 1D). Consistent with the findings of others (6), we detected Grb14 expression in fat, muscle, and liver, and peripheral disruption of Grb10 had no effect on Grb14 expression in these tissues (Fig. 1D). Disruption of Grb10 also had no significant effect on the expression of other insulin molecules, such as p44/42 MAPK (Fig. 1C and D).
Effects of peripheral disruption of Grb10 on body weight and body composition. Disruption of Grb10 gene expression was previously shown to cause increased animal size at birth (4). However, it was undetermined whether disruption of Grb10 affected growth in adult mice. To determine the role of Grb10 in regulating growth in the adult mouse, we measured the body weight and body size of both wild-type and Grb10-deficient mice weekly. Grb10-deficient male mice displayed greater body weight (Fig. 2A) and body size (Fig. 2B) than their wild-type littermates. Similar findings were observed in female mice (data not shown). Using DEXA, we found that peripheral disruption of Grb10 led to a significant increase in fat and lean body mass in both male and female mice (Fig. 3A and data not shown). However, this increase in lean body mass, which primarily represents muscle, was proportional to the increase in body size in Grb10-deficient mice (Fig. 3B). We also examined whether the overgrowth was associated with a proportional overgrowth of other mouse tissues. In some tissues (kidney, heart, spleen, and brain) the weights were similar in wild-type and Grb10-deficient mice (Fig. 3C). However, the weights of several tissues (muscle, pancreas, and lung) were significantly increased in the Grb10-deficient mice compared to wild-type littermates (Fig. 3C). A similar increase in organ weights also was observed in female mice (data not shown).
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7 µg/liter) in the wild-type and Grb10-deficient groups during the last 30 min of the clamp (data not shown). On the other hand, the insulin-stimulated Rd, a reflection of muscle insulin sensitivity, was significantly higher in the Grb10-deficient mice than in wild-type littermate controls fed with a regular chow diet (Fig. 4C). Targeted deletion of the Grb10 gene also blunted HFD-induced insulin resistance (Fig. 4D). Because the insulin infusion employed in the present study caused a near-maximal suppression of HGP in chow-fed mice, the glucose infusion rate closely mirrored the Rd and also was significantly higher in the Grb10-deficient mice, consistent with the tracer turnover data, which demonstrate that Grb10-deficient mice have increased insulin sensitivity compared with wild-type controls (Fig. 4E and F). On the other hand, there was no significant difference in the insulin-mediated suppression of HGP during the clamp (a measure of liver insulin sensitivity) between Grb10-deficient mice and their wild-type littermates fed with regular chow (Fig. 4G). While insulin-mediated suppression of HGP was reduced by HFD in both Grb10-deficient mice and their wild-type littermates, no significant difference was observed in the insulin-mediated suppression of HGP between these two groups (Fig. 4H), consistent with the idea that Grb10 does not play a significant role in regulating liver insulin sensitivity. These findings demonstrate that Grb10-deficient mice have increased insulin sensitivity in vivo and that the main site responsible for this difference is muscle, the primary site of glucose disposal under hyperinsulinemic conditions.
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DISCUSSION |
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Based upon the observation that suppression of endogenous Grb10 levels by RNA interference led to enhanced insulin/IGF-1 signaling in cells (9, 13), we hypothesized that a deficiency of Grb10 in insulin target tissues in mice would result in enhanced insulin signaling and sensitivity in vivo. Consistent with this hypothesis, we found that insulin produced a greater stimulation of Akt phosphorylation in muscle and fat of Grb10-deficient mice than in their wild-type littermates (Fig. 5A to C). We also measured the insulin sensitivity of the Grb10-deficient mice and their wild-type littermates using the gold standard hyperinsulinemic-euglycemic clamp. Based on the finding that Grb10 is absent in adult liver and adult hepatocytes (10) (Fig. 1C), we anticipated that the Grb10 knockout would not have a significant effect on HGP. Thus, we employed an insulin infusion rate (18 mU/kg·min) that caused a near-maximal suppression of HGP in chow-fed mice in order to more clearly define the effect of Grb10 on insulin-stimulated glucose uptake in muscle. Our studies revealed that Grb10-deficient mice had a significantly higher rate of insulin-stimulated whole-body (primarily reflecting muscle) glucose disposal than their wild-type littermates (Fig. 4C to F). Since both the muscle mass and the total mass were increased in the Grb10 knockout mice, the enhanced Rd could be due to increased muscle mass. However, the muscle mass/total mass ratio was not significantly different between the wild-type and Grb10-knockout mice (Fig. 3B). Thus, the finding that the Rd per kg body mass is increased in the Grb10 knockout mice suggests a direct effect of disrupting Grb10 gene expression on improved muscle insulin sensitivity. Peripheral Grb10 deficiency had little effect on insulin-stimulated Akt phosphorylation in the liver (data not shown) and insulin-mediated suppression of HGP (Fig. 4G and H).
In addition to regulating insulin signaling and glucose homeostasis, Grb10 also has been shown to play a role in regulating the growth of both the embryo and the placenta (4). To determine whether Grb10 plays a role in regulating postnatal growth, we compared the body weights and body sizes of Grb10-deficient mice and their littermates. Peripheral disruption of the Grb10 gene led to increased body weight and animal size (Fig. 2 and data not shown). These results, along with those of Shiura et al. (23), who showed that overexpression of Grb10 leads to growth retardation in adult mice, support a role for Grb10 as a negative regulator of growth in adult as well as in embryonic mice. Consistent with the findings that Grb10 is highly expressed in the pancreas and moderately expressed in the lung and muscle, disruption of Grb10 gene expression in these tissues led to enhanced weights of these tissues (Fig. 3C). On the other hand, little difference was observed in the liver and brain weights of Grb10-deficient mice and their littermates (Fig. 3C), consistent with the findings that the expression levels of Grb10 were not affected in these tissues in wild-type and Grb10-deficient mice (Fig. 1C).
Grb10 belongs to the family that also includes Grb7 and Grb14. These proteins share a conserved molecular architecture, including the PH domain, the BPS region, and the SH2 domain, and these display some overlap in function (12). In mice, Grb7 is highly expressed in kidney cells but in smaller amounts in ovaries, testes, and lung tissues (22). Grb14 is also highly expressed in mouse liver, but its expression in fat and muscle is moderate and low, respectively (3). While the in vivo role of Grb7 in the regulation of energy homeostasis remains largely unknown, targeted deletion of the Grb14 gene has been shown to improve insulin sensitivity and glucose homeostasis, suggesting that this protein functions in vivo as a negative regulator of insulin signaling and action (6). The findings that Grb10 and Grb14 are expressed specifically in different insulin-sensitive tissues and that disruption of their expression leads to enhanced insulin sensitivity suggest that these two IR binding proteins may regulate insulin signaling and action in a tissue-specific manner. Since disruption of the Grb10 gene had no effect on either Grb7 or Grb14 expression (Fig. 1D), the increase in muscle insulin sensitivity observed in the present study cannot be explained by compensatory changes in the expression levels of the other two Grb7/10/14 family members.
The binding of insulin to its membrane receptor activates two major pathways, the PI 3-kinase/Akt pathway and the MAPK signaling pathway. Activation of the PI 3-kinase/Akt and MAPK signaling pathways plays an essential role in the regulation of insulin-mediated metabolic events, such as uptake and subsequent storage of glucose, and mitogenic events, such as DNA replication, cell growth, and division (24). The signaling specificity of the IR is regulated through multiple mechanisms, including ligand concentration, duration of stimulation, and the dissociation rate of ligand from the receptor (24). We found that i.p. injection of insulin at a lower dose (1 U/kg body weight) for 5 min led to greater Akt phosphorylation in both muscle and fat of the Grb10-deficient mice (Fig. 5A and B). Little difference in insulin-stimulated MAPK phosphorylation could be observed between Grb10-deficient mice and their wild-type littermates under these conditions (data not shown). Interestingly, a slight increase in muscle Akt phosphorylation was also detected in the Grb10-deficient mice under fasting conditions (Fig. 5A). However, statistical analysis revealed no significant difference in basal Akt phosphorylation between Grb10-deficient mice and their wild-type littermates. I.p. injection of insulin at a higher dose (5 U/kg body weight) for a longer time (10 min) led to a marked increase in MAPK phosphorylation in both the Grb10-deficient mice and their wild-type littermates, and the response in the Grb10-deficient mice was significantly higher than in the wild-type mice (Fig. 5D and E). Under these conditions, however, no significant difference in Akt phosphorylation was observed between wild-type and Grb10-deficient mice (Fig. 5F and G). These findings suggest that the higher dose and longer time of insulin administration may cause Akt phosphorylation to reach a plateau, and therefore, the regulatory effect of Grb10 on insulin-stimulated Akt phosphorylation is masked. This result is interesting because it suggests that the metabolic and mitogenic effects of insulin may be differentially regulated in vivo. Consistent with this, it has been shown that longer association of insulin with its receptor, which may result from a higher insulin concentration and prolonged presence of insulin, correlates with enhanced mitogenic signaling (24). However, it should be noted that the insulin dose we used for i.p. injection was more than 50-fold higher than that used in the insulin clamp study (1 U/kg or 5 U/kg versus 18 mU/min·kg). In addition, a single dose of insulin was used in the i.p. injection experiments, whereas insulin was continuously infused during the clamp study. Therefore, the time course and dose-response curve were different in i.p. injection and insulin clamp experiments. Consistent with this, we found that Akt phosphorylation was greatly increased in muscle of the Grb10-deficient mice collected at the end of the insulin clamp study compared to wild-type littermates (Fig. 5C).
We recently found that suppression of Grb10 by RNA interference led to increased stability of the IRß in HeLa cells overexpressing the IR (20). To examine whether knocking out Grb10 has any effect on the expression level of IRß in vivo, we examined the expression levels of IRß in muscle and fat tissues of the Grb10-deficient mice and their wild-type littermates. No significant difference in the expression levels of IRß was observed in tissues between wild-type and Grb10-deficient mice fed with normal chow or HFD (data not shown), suggesting that Grb10 may not play a role in IRß stability in vivo or that the negative effect of Grb10 on IRß stability observed in vitro (20) may be masked by some compensatory mechanisms in vivo. Alternatively, the effect of Grb10 on IRß stability may be induced only by prolonged insulin stimulation.
In summary, we showed that disruption of Grb10 gene expression in mice results in enhanced insulin signaling through the PI 3-kinase and MAPK pathways in association with increased muscle insulin sensitivity. In addition, the loss of Grb10 expression in peripheral tissues led to overgrowth in adult mice, demonstrating that endogenous Grb10 is a growth suppressant in adult, as well as embryonic, mice. Our results provide strong evidence that endogenous Grb10 functions as a negative regulator of insulin signaling and action in vivo.
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ACKNOWLEDGMENTS |
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We thank Vivian Diaz and her staff at the Nathan Shock Animal Core for their expert care of the mice.
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FOOTNOTES |
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Published ahead of print on 9 July 2007.
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