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Departments of 1Cellular and Molecular Medicine and 2Respirology, Graduate School of Medicine, Chiba University, Chiba 260-8670; 3Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507; 4Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655; and 5Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
Submitted 19 June 2003 ; accepted in final form 11 August 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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insulin receptor substrate-1; insulin secretion; insulin sensitivity; pancreatic 
-cell; skeletal muscle
-cell KATP channel comprises Kir6.2 and SUR1 (12, 23), and the cardiac and skeletal muscle KATP channels comprise Kir6.2 and SUR2A (1, 4). In pancreatic -cells, glucose is transported through the glucose transporter GLUT2, and the subsequent metabolism of glucose produces ATP. The increase in the ATP/ADP ratio closes the KATP channels, depolarizing the -cell membrane and opening the voltage-dependent calcium channels to allow calcium influx. The rise in intracellular calcium concentration triggers insulin granule exocytosis. Because there is almost no insulin secretion in response to glucose in mice lacking Kir6.2 (18), the pancreatic -cell KATP channel clearly plays a critical role in glucose-induced insulin secretion. Although Kir6.2 knockout mice lack glucose-induced insulin secretion, they exhibit only slight glucose intolerance and do not develop overt diabetes (18). Furthermore, an insulin tolerance test shows enhanced insulin action in Kir6.2 knockout mice (18). We (17) found recently that glucose uptake in skeletal muscles of Kir6.2 knockout mice is increased both in vivo and in vitro. Chutkow et al. (7) also reported that disruption of KATP channels enhances glucose uptake in skeletal muscle. In the present study, we crossed Kir6.2 knockout mice with insulin receptor substrate-1 (IRS-1) knockout mice to clarify the mechanism of KATP channel-mediated glucose uptake. IRS-1 knockout mice exhibit insulin resistance in skeletal muscles (3, 30, 35). However, double-knockout mice with disruption of both the Kir6.2 and IRS-1 genes do not develop insulin resistance or overt diabetes. Disruption of Kir6.2 does not increase phosphatidylinositol 3-kinase (PI3K) activity or Akt/PKB phosphorylation, indicating that KATP channel-mediated enhancement of glucose uptake is not mediated by IRS-1/PI3K signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Oral glucose tolerance test and insulin tolerance test. An oral glucose tolerance test (OGTT) was performed on male mice fasted for 16 h at 12–20 wk of age, and glucose (1.5 g/kg) was administered orally with a feeding needle. Blood samples were collected before and after glucose loading at the time points indicated in the figures. Blood glucose levels were measured in whole blood with an Autosense Glucose II glucose analyzer (Sankyo, Tokyo, Japan). Area under the curve (AUC) was calculated for glucose during OGTT. In the insulin tolerance test (ITT), human insulin (0.5 U/kg) was injected intraperitoneally in conscious adult mice fed ad libitum at 12–20 wk of age. Serum insulin levels were determined with an ELISA kit (Morinaga, Yokohama, Japan). Insulin and glucose responses during the OGTT were calculated as the incremental serum insulin values (
I) integrated over the period (30 or 60 min) and the corresponding incremental integrated blood glucose values (G). The insulinogenic index (I/G) (11) represents the insulin response relative to the blood glucose concentration.
Measurement of rate constant of net uptake of 2-deoxy-D[3H]glucose. Each mouse after an overnight fast (16 h) was injected through a tail vein with 12.5 µCi of 2-deoxy-D[3H]glucose (2-[3H]DG; 33.0 Ci/mmol) and 2.5 µCi of [U-14C]sucrose (565 mCi/mmol) (Amersham Pharmacia Biotech, Uppsala, Sweden) dissolved in saline solution. Blood samples were taken before and 2, 5, and 10 min after the injection. Plasma samples were analyzed for radioactivity of 2-[3H]DG and [14C]sucrose. Immediately after final blood sampling, the mice were killed by cervical dislocation. Skeletal muscles [gastrocnemius, soleus, and extensor digitorum longus (EDL)] and white adipose tissues (epididymal and retroperitoneal fat) were rapidly dissected and weighed. The rate constant (Ki) of net tissue uptake of 2-[3H]DG was calculated as described previously (29).
Protein analysis. For immunoblot analysis, the mice were anesthetized with pentobarbital sodium (50 mg/kg ip), and gastrocnemius muscles were removed. Tissues were homogenized in a buffer composed of 25 mM Tris · HCl (pH 7.4), 10 mM Na3VO4, 10 mM NaF, 10 mM Na4P2O7, 10 mM EDTA, 10 mM EGTA, 1% Nonidet P-40, 1 mM PMSF, with protease inhibitor cocktail (Sigma, St. Louis, MO), and centrifuged at 15,000 g at 4°C for 30 min to remove insoluble materials. Supernatants containing equal amounts of protein (40 µg) were subjected to SDS-PAGE and transferred to Immobilon P membranes (Millipore, Bedford, MA). The membranes were probed with antibody raised against GLUT4 (Chemicon International, Temecula, CA), Akt/PKB, phosphorylated Akt/PKB (Thr308 or Ser473), cAMP-activated protein kinase (AMPK), or phosphorylated AMPK (Thr172) (Cell Signaling Technology, Beverly, MA) and visualized with enhanced chemiluminescence (Amersham Pharmacia Biotech).
For measurement of PI3K activity, gastrocnemius muscles were removed from anesthetized mice 5 min after insulin (5 U/mouse) or vehicle administration through the vena cava. Tissue lysates prepared as described above were subjected to immunoprecipitation with anti-phosphotyrosine antibody (PY20; Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitates were immobilized on protein A-agarose beads (Pierce Biotechnology, Rockford, IL) followed by an extensive wash with the tissue homogenization buffer and then suspended in 50 µl of PI3K reaction buffer composed of 20 mM Tris · HCl (pH 7.4), 100 mM NaCl, and 0.5 mM EGTA containing 0.1 mg/ml of phosphatidylinositol. Reactions were initiated by adding 5 µl of a MgCl2-ATP mixture (200 mM MgCl2, 200 µM ATP) containing 5 µCi of [
-32P]ATP to the immunoprecipitates and incubated at 25°C for 20 min. Reactions were terminated by adding 20 µl of 8 N HCl and 160 µl of chloroform-methanol (1:1). The organic phase was spotted onto a silica gel plate (Merck, Darmstadt, Germany) and developed in CHCl3:CH3OH:NH4OH(28%):H2O (43:38:5:57). The phosphorylated lipids were visualized by autoradiography. Signals were quantified by scanning densitometry (Amersham Pharmacia Biotech).
Statistical analysis. Values are expressed as means ± SE. The significance of differences between test groups was evaluated by t-test or by one-way analysis of variance followed by Scheffé's test. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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70% of WT. Kir6.2–/–IRS-1–/– also showed growth retardation similar to that of Kir6.2+/+IRS-1–/– (Table 1).
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Blood glucose and insulin levels in ad libitum-fed and fasted mice. To evaluate the effects of double disruption of both the Kir6.2 and IRS-1 genes on glucose metabolism in vivo, we measured blood glucose and serum insulin levels fed ad libitum and after 16 h of fasting (Table 1). Fed glucose levels were similar among the four groups of mice. As in a previous study (3), fed insulin levels of Kir6.2+/+IRS-1–/– were increased significantly compared with WT (P = 0.0075). In addition, Kir6.2–/–IRS-1–/– showed significantly higher fed insulin levels than Kir6.2–/–IRS-1+/+ (P < 0.0001). In the fasted state, Kir6.2+/+IRS-1–/– had blood glucose levels comparable to those of WT (P = 0.3741), whereas Kir6.2–/–IRS-1+/+ and Kir6.2–/–IRS-1 –/– had lower blood glucose levels (P = 0.0003 for Kir6.2–/–IRS-1+/+ vs. WT and P = 0.0002 for Kir6.2–/– IRS-1–/– vs. WT). Although the fasting insulin levels of Kir6.2+/+IRS-1–/– and WT were similar (P = 0.0681), the levels of Kir6.2–/–IRS-1–/– were significantly increased compared with those of Kir6.2–/–IRS-1+/+ (P = 0.0150). These results indicate that lack of IRS-1 induces hyperinsulinemia, especially in the fed state, without affecting blood glucose levels, whereas disruption of Kir6.2 decreases fasting blood glucose levels without affecting serum insulin levels. Overt diabetes did not develop in mice with disruption of both Kir6.2 and IRS-1.
Blood glucose and serum insulin levels during OGTT. We then compared OGTT (1.5 g/kg) in the four groups of mice (Fig. 1) to clarify the effects of double disruption of the Kir6.2 and IRS-1 genes on acute glucose metabolism. As reported previously (18, 30), Kir6.2+/+IRS-1–/– have normal glucose tolerance, whereas Kir6.2–/–IRS-1+/+ have impaired glucose tolerance (Fig. 1A). The AUC of blood glucose is largest in Kir6.2–/–IRS-1+/+ among the four groups (Fig. 1B). Surprisingly, the impaired glucose tolerance observed in Kir6.2–/–IRS-1+/+ was not further impaired by disruption of IRS-1 (in Kir6.2–/–IRS-1–/–), and the glucose disappearance rate from peak at 30 min was enhanced compared with Kir6.2–/–IRS-1+/+ (Fig. 1A). The AUC for Kir6.2–/–IRS-1–/– was similar to that for WT (Fig. 1B), also indicating that glucose tolerance is not deteriorated in Kir6.2–/–IRS-1–/–. Serum insulin levels during OGTT are shown in Fig. 1C. The insulin secretion in Kir6.2–/–IRS-1+/+ did not respond to glucose challenge, and Kir6.2+/+IRS-1–/– exhibited enhanced insulin response, as reported previously (18, 32). The insulin response in Kir6.2–/–IRS-1–/– was poor at 30 min (P = 0.4580 vs. at 0 min) but was significantly enhanced at 60 min (P = 0.0266 vs. at 0 min). Insulin levels in Kir6.2–/–IRS-1–/– were always higher than in Kir6.2–/–IRS-1+/+ during the testing period. The insulinogenic index (I/G) shows that glucose-induced insulin secretion is enhanced in Kir6.2+/+IRS-1–/– but diminished in Kir6.2–/–IRS-1 +/+ (Fig. 1D). Kir6.2–/–IRS-1–/– also had a lower I/G than WT but showed a higher index than Kir6.2–/–IRS-1+/+ during 0–60 min (P = 0.0486). These observations show that the acute insulin response is impaired by lack of the Kir6.2 gene, whereas disruption of the IRS-1 gene partially restores glucose-induced insulin secretion even in the absence of the Kir6.2 gene.
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Assessment of insulin sensitivity in vivo. We then assessed insulin sensitivity by ITT (0.5 U/kg ip). In accord with previous data (18, 30), the drop in blood glucose level after insulin injection was less in Kir6.2+/+IRS-1–/– than in WT, whereas it was significantly greater in Kir6.2–/–IRS-1+/+ (Fig. 2). The blood glucose level decrease in Kir6.2–/–IRS-1–/– was more than in Kir6.2+/+IRS-1–/– and in WT but similar to that in Kir6.2–/–IRS-1+/+ (Fig. 2). These results show that disruption of the Kir6.2 gene leads to enhancement of insulin sensitivity even in the absence of the IRS-1 gene.
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2-[3H]DG uptake in skeletal muscles and fat tissues. We previously found that 2-[3H]DG uptake in skeletal muscles of Kir6.2 knockout mice is enhanced (17). Here, we measured 2-[3H]DG uptake to determine whether disruption of Kir6.2 increases glucose uptake in the absence of IRS-1. In skeletal muscles [gastrocnemius, soleus, and extensor digitorum longus (EDL)], the rate constant of net uptake of 2-[3H]DG was similar in Kir6.2+/+IRS-1–/– and WT (Fig. 3A). However, in Kir6.2–/–IRS-1+/+, the 2-[3H]DG uptake rates in gastrocnemius and EDL were significantly higher than in WT (Fig. 3A). Moreover, 2-[3H]DG uptake in Kir6.2–/– IRS-1–/– was enhanced in gastrocnemius compared with Kir6.2+/+IRS-1–/–, demonstrating that disruption of the Kir6.2 gene enhances glucose uptake even in the absence of the IRS-1 gene. In contrast, the rate constant of net uptake of 2-[3H]DG in white adipose tissues (epididymal and retroperitoneal fat) was higher in Kir6.2+/+IRS-1–/– than in WT (Fig. 3B). 2-[3H]DG uptake in retroperitoneal fat also was increased in Kir6.2–/–IRS-1–/– compared with WT (Fig. 3B).
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Expression of GLUT1 and GLUT4 in skeletal muscle. To test the possibility that change in expression of glucose transport proteins is involved in Kir6.2-mediated glucose uptake, we performed immunoblotting of GLUT1 and GLUT4 proteins. No significant difference in levels of GLUT4 protein was detected in skeletal muscles among all four groups of mice (Fig. 4). GLUT1 protein was not detected by immunoblotting in any of the groups (data not shown). Therefore, it is unlikely that enhanced glucose uptake in Kir6.2–/–IRS-1+/+ and Kir6.2–/–IRS-1–/– is due to upregulation of GLUT1 or GLUT4 expression.
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PI3K activity in skeletal muscle. Because the metabolic action of insulin is mediated by activation of PI3K (22), we compared skeletal muscle PI3K activity with and without insulin injection. In the presence of exogenous insulin, no significant difference in PI3K activity was observed among four groups of mice (Fig. 5A; P values are indicated in the figure). However, the fold increase in PI3K activity after insulin injection was significantly lower in Kir6.2+/+IRS-1–/– than in WT (Fig. 5B), indicating that insulin action is diminished by the lack of IRS-1. The difference in the fold increase between Kir6.2–/–IRS-1+/+ and WT was not significant (P = 0.0869). In addition, the fold increases in PI3K activity were similar in Kir6.2+/+IRS-1–/– and Kir6.2–/–IRS-1–/– (P = 0.7675), suggesting that disruption of the Kir6.2 gene does not affect the PI3K signal. The fold increases in PI3K activity in Kir6.2–/– IRS-1+/+ and Kir6.2–/–IRS-1–/– were comparable (P = 0.9607), indicating that lack of the IRS-1 gene does not affect insulin action when the Kir6.2 gene is disrupted.
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Phosphorylation of Akt/PKB in skeletal muscle. Akt/PKB is one of the signaling molecules downstream of PI3K that regulates glucose uptake in skeletal muscle. It is activated by phosphorylation at Thr308 and Ser473 (34). The abundance of Akt/PKB proteins in all four groups of mice is similar (Fig. 6A). In addition, similar insulin-induced phosphorylation (at both Thr307 and Ser473) of Akt/PKB is detected among the four groups of mice (Fig. 6, B and C), demonstrating that disruption of the IRS-1 or the Kir6.2 gene does not affect Akt/PKB phosphorylation.
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AMPK in skeletal muscle. Because AMPK is known to regulate insulin-independent glucose uptake in skeletal muscle (16), we investigated protein expression and phosphorylation of AMPK. Protein expression of AMPK in skeletal muscle of Kir6.2+/+IRS-1–/– was decreased significantly compared with WT (P = 0.0452; Fig. 7A). In Kir6.2–/–IRS-1+/+ and Kir6.2–/–IRS-1–/–, AMPK protein levels showed no significant change (P = 0.1542 for Kir6.2–/–IRS-1+/+ vs. WT and P = 0.1184 for Kir6.2–/–IRS-1–/– vs. WT). Phosphorylation of AMPK (an activated form) is shown in Fig. 7B. Although Kir6.2+/+IRS-1–/– have levels of phosphorylated AMPK similar to those of WT (P = 0.3065), both Kir6.2–/–IRS-1+/+ and Kir6.2–/–IRS-1–/– had levels significantly lower than in WT (P = 0.0183 for Kir6.2–/– IRS-1+/+ vs. WT and P = 0.0101 for Kir6.2–/– IRS-1–/– vs. WT). These results show that lack of the IRS-1 gene reduces protein expression of AMPK in skeletal muscle and that lack of the Kir6.2 gene reduces the phosphorylated form of AMPK.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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IRS-1-mediated glucose uptake requires activation of PI3K followed by activation of downstream signals such as atypical PKCs and Akt/PKB (6, 10, 14, 27, 31, 33). Accordingly, disruption of the IRS-1 gene causes reduction of insulin-stimulated activation of PI3K (Refs. 3 and 30 and Fig. 5B). We also found that lack of the Kir6.2 gene does not increase PI3K activity in either the presence or the absence of the IRS-1 gene (Fig. 5B), indicating that a Kir6.2-associated increase in glucose uptake is independent of PI3K activity. Furthermore, the phosphorylation of Akt/PKB and the amounts of the protein are not affected by the lack of the Kir6.2 gene, indicating that the Akt/PKB signal is not involved in Kir6.2-associated increases in glucose uptake.
Glucose uptake in skeletal muscles also is stimulated by an insulin-independent, AMPK-dependent mechanism (19, 20). Upon phosphorylation, AMPK stimulates glucose uptake additively to insulin action. Accordingly, if the AMPK signal is enhanced, glucose uptake should be increased. However, this was not the case in Kir6.2 knockout mice, as the phosphorylated form of AMPK was not increased but decreased in mice lacking the Kir6.2 gene (Fig. 7B). Although the pathways involved in KATP channel-mediated glucose uptake remain to be elucidated, the present results establish that they are independent of AMPK and the IRS-1/PI3K signal.
The acute insulin response was evaluated by OGTT (Fig. 1C). Although disruption of the Kir6.2 gene leads to impaired glucose-induced insulin secretion, lack of the IRS-1 gene causes hypersecretion. Double-knockout mice have a poor insulin response at 30 min but a significant response at 60 min, which may account for the acceleration of the disappearance rate of blood glucose after 30 min compared with Kir6.2 knockout mice. Thus disruption of IRS-1 gene partially restores glucose-induced insulin secretion, even in the absence of Kir6.2 gene. Islet insulin content of double-knockout mice was decreased to 84% of that of WT mice (data not shown), similarly to that of IRS-1 knockout mice (15). Kir6.2 knockout islets showed insulin content similar to that of WT islets (data not shown). Glucose-induced insulin secretion in Kir6.2 knockout islets is severely impaired (18). In addition, IRS-1 knockout mice exhibit -cell hyperplasia, whereas insulin secretion from the isolated islets is diminished (15, 32). Thus it is likely that the increased insulin response in the double-knockout mice is due to -cell hyperplasia rather than to enhanced insulin secretion from individual -cells.
Despite impaired acute insulin secretion, fed insulin levels are increased in double-knockout mice similarly to those of IRS-1 knockout mice. IRS-1 knockout mice have -cell hyperplasia (15, 32), which may account for the hyperinsulinemia. In general, -cell hyperplasia and the resulting hyperinsulinemia are thought to be a compensatory response to insulin resistance. However, the double-knockout mice in the present study have no insulin resistance, as assessed by ITT (Fig. 2). Therefore, the hyperinsulinemia observed in these mice is not due to insulin resistance, and the lack of IRS-1 can lead to hyperinsulinemia independently of the onset of insulin resistance, at least in the absence of Kir6.2.
In conclusion, analysis of mice with disruption of both the Kir6.2 and IRS-1 genes reveals that KATP channel-associated enhancement of glucose uptake in skeletal muscles is independent of the IRS-1/PI3K-signaling pathway. In addition, these double-knockout mice provide a useful model to study the interaction of -cell function and peripheral glucose metabolism.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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), Kir6.2–/– (
), IRS-1–/– (
), and DK (
) fed ad libitum. Blood samples were obtained at indicated time points. Blood glucose concentration was determined as described in MATERIALS AND METHODS. Data are percentages of initial value (mean ± SE; n = 7–12 animals). *P < 0.05, **P < 0.01 compared with WT; ##P < 0.01 compared with Kir6.2–/–.
