ATP-sensitive K+ channel-mediated glucose uptake is independent of IRS-1/phosphatidylinositol 3-kinase signaling
Kohtaro Minami,1,3
Mizuo Morita,1,2
Atsunori Saraya,1
Hideki Yano,1
Yasuo Terauchi,4
Takashi Miki,1
Takayuki Kuriyama,2
Takashi Kadowaki,4 and
Susumu Seino1,5
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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We previously found that disruption of Kir6.2-containing ATP-sensitive K+ (KATP) channels increases glucose uptake in skeletal muscle, but the mechanism is not clear. In the present study, we generated knockout mice lacking both Kir6.2 and insulin receptor substrate-1 (IRS-1). Because IRS-1 is the major substrate of insulin receptor kinase, we expected disruption of the IRS-1 gene to reduce glucose uptake in Kir6.2 knockout mice. However, the double-knockout mice do not develop insulin resistance or glucose intolerance. An insulin tolerance test reveals the glucose-lowering effect of exogenous insulin in double-knockout mice and in Kir6.2 knockout mice to be similarly enhanced compared with wild-type mice. The basal 2-deoxyglucose uptake rate in skeletal muscle of double-knockout mice is increased similarly to the rate in Kir6.2 knockout mice. Accordingly, disruption of the IRS-1 gene affects neither systemic insulin sensitivity nor glucose uptake in skeletal muscles of Kir6.2-deficient mice. In addition, no significant changes were observed in phosphatidylinositol 3-kinase (PI3K) activity and its downstream signal in skeletal muscle due to lack of the Kir6.2 gene. Disruption of Kir6.2-containing KATP channels clearly protects against IRS-1-associated insulin resistance by increasing glucose uptake in skeletal muscles by a mechanism separate from the IRS-1/PI3K pathway.
insulin receptor substrate-1; insulin secretion; insulin sensitivity; pancreatic
-cell; skeletal muscle
ATP-SENSITIVE K+ (KATP) CHANNELS regulate various cellular functions such as secretion and neural excitability by coupling cell metabolism to membrane potential (2, 5, 9, 21, 25, 26, 28). The KATP channel is a heterooctamer composed of four pore-forming subunits, Kir6.1 or Kir6.2, and four sulfonylurea receptor subunits, SUR1, SUR2A, or SUR2B (8, 13). Differing combinations of Kir6.1 or Kir6.2 and SUR1, SUR2A, or SUR2B constitute KATP channels with distinct nucleotide and pharmacological sensitivities (24). For example, the pancreatic
-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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Generation of double-knockout mice. Homozygous mutant mice for the Kir6.2 gene (Kir6.2/) (18) and for the IRS-1 gene (IRS-1/) were generated as described previously (30). To generate double-knockout mice, Kir6.2/ were crossbred with IRS-1/. To homogenize the genetic background, the F1 generation was used for multiple breedings to obtain the F2 generation, which comprised nine different genotypes on a similar mixed genetic background: Kir6.2+/+IRS-1+/+, Kir6.2+/+IRS-1+/, Kir6.2+/+IRS-1/, Kir6.2+/IRS-1+/+, Kir6.2+/IRS-1+/, Kir6.2+/IRS-1/, Kir6.2/IRS-1+/+, Kir6.2/IRS-1+/, and Kir6.2/IRS-1/. Among the nine genotypes generated, mice with the following four genotypes were used for the present studies: Kir6.2+/+IRS-1+/+ (wild-type, WT), Kir6.2/IRS-1+/+, Kir6.2+/+IRS-1/, and Kir6.2/IRS-1/. The genotypes were confirmed by PCR, as previously described (18, 30). The mice were fed normal chow and maintained using standard husbandry procedures. Animal care and procedures were approved by the Animal Care Committee of Chiba University School of Medicine.
Oral glucose tolerance test and insulin tolerance test. An oral glucose tolerance test (OGTT) was performed on male mice fasted for 16 h at 1220 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 1220 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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General features of the double-knockout mice. Double-knockout mice (Kir6.2/IRS-1/) were generated by breeding Kir6.2/ and IRS-1/. Each genotype of mice was identified using the PCR method. WT, Kir6.2/IRS-1+/+, Kir6.2+/+IRS-1/, and Kir6.2/ IRS-1/ were born at the expected Mendelian frequency. Although the growth of Kir6.2/IRS-1+/+ was similar to that of WT, Kir6.2+/+IRS-1/ showed growth retardation, as previously reported (18, 30). The body weight of Kir6.2+/+IRS-1/ was
70% of WT. Kir6.2/IRS-1/ also showed growth retardation similar to that of Kir6.2+/+IRS-1/ (Table 1).
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 060 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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Fig. 1. Glucose and insulin responses during oral glucose tolerance test. Wild-type (WT), Kir6.2/IRS-1+/+ (Kir6.2/), Kir6.2+/+IRS-1 / (IRS-1/), and Kir6.2/IRS-1/ (DK) mice (1220 wk of age) were fasted overnight (16 h) before oral glucose loading (1.5 g/kg body wt). Blood samples were taken at indicated time points. Concentrations of blood glucose and serum insulin were determined as described inMATERIALS AND METHODS. Blood glucose levels (A), area under the curves (AUC) for blood glucose (B), serum insulin levels (C), and insulinogenic indexes (D) of WT, Kir6.2/, IRS-1/, and Kir6.2/IRS-1/ [double knockout (DK)] are shown with SE bars (n). The insulinogenic index ( I/ G) represents the ratio of the integrated incremental serum insulin values ( I, pmol/l) and integrated incremental blood glucose values ( G, mmol/l). *P < 0.05, **P < 0.01 compared with WT; #P < 0.05, ##P < 0.01 compared with Kir6.2/.
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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.
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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Fig. 3. Rate constants (Ki) of 2-deoxy-[3H]glucose (2-[3H]DG) uptake in skeletal muscle (A) and adipose tissue (B). Mice (1220 wk) fasted overnight (16 h) were injected with 12.5 µCi of 2-[3H]DG and 2.5 µCi of [U-14C]sucrose via tail vein. Immediately after final blood sampling, skeletal muscles and adipose tissues were dissected and weighed. Ki of net tissue uptake of 2-[3H]DG was calculated as described previously (24). Data are means ± SE (n, no. of animals). *P < 0.05, **P < 0.01 compared with WT; #P < 0.05 compared with Kir6.2/.
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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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Fig. 4. Immunoblot analyses of GLUT4. Equal amounts of tissue lysates (40 µg) were separated by 7.5% SDS-PAGE, probed with anti-GLUT4 antibody, and visualized with enhanced chemiluminescence. Bands were quantified with a densitometer, and each bar represents mean ± SE (n). Representative photograph is shown. Difference between WT and each group was not statistically significant.
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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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Fig. 5. Phosphatidylinositol 3-kinase (PI3K) activity in the presence or absence of insulin injection (A) and fold increase of its activity after insulin injection (B) in skeletal muscle. After treatment with or without insulin for 5 min, gastrocnemius lysates were immunoprecipitated with anti-phosphotyrosine antibody and then subjected to PI3K assay as described in MATERIALS AND METHODS. A: results are expressed as percent WT without insulin, and each bar represents mean ± SE (n). Difference between WT and each group was not statistically significant. Photograph shows representative results. B: results are expressed as ratio of PI3K activity with and without insulin, and each bar represents mean ± SE (n). *P < 0.05. NS, not significant.
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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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Fig. 6. Immunoblotting of Akt/PKB (A), Thr308 phosphorylated (p-)Akt/PKB (B), and Ser473 p-Akt/PKB (C). The gastrocnemius lysates (40 µg) were subjected to SDS-PAGE and transferred to Immobilon P membranes. The membranes were probed with anti-Akt/PKB or anti-p-Akt/PKB (Thr308 or Ser473) as described in MATERIALS AND METHODS. Photographs show representative results. A: Akt/PKB protein expression levels of all 4 groups of mice are similar (n = 3). B and C: results are expressed as percent WT with insulin, and each bar represents mean ± SE (n). Difference between WT and each group is not statistically significant.
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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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Fig. 7. Immunoblotting of cAMP-activated protein kinase (AMPK; A) and phosphorylated AMPK (B). Gastrocnemius lysates (40 µg) were subjected to SDS-PAGE and transferred to Immobilon P membranes. Membranes were probed with anti-AMPK or anti-phospho-AMPK (Thr172) as described in MATERIALS AND METHODS. Photographs show representative results. Results are expressed as percent WT, and each bar represents mean ± SE (n). *P < 0.05. NS, not significant.
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DISCUSSION
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Double-knockout mice with disruption of both the Kir6.2 and IRS-1 genes show no overt diabetes and no insulin resistance. We previously found that Kir6.2-deficient mice exhibit enhanced insulin sensitivity (18) and that Kir6.2-containing KATP channels participate in glucose uptake in skeletal muscles (17), but the mechanism was unclear. Because IRS-1 is a major substrate of insulin receptor kinase and mediates downstream insulin signals, lack of the IRS-1 gene causes insulin resistance (30). Accordingly, a KATP channel-associated increase in insulin action or glucose uptake would be mediated by the IRS-1 signal, so disruption of the IRS-1 gene should reduce both insulin sensitivity and glucose metabolism in Kir6.2 knockout mice. Lack of the Kir6.2 gene should not affect insulin action or glucose metabolism when the IRS-1 gene is disrupted. However, the present study demonstrates that disruption of the Kir6.2 gene enhances insulin sensitivity and glucose metabolism in the absence of the IRS-1 gene and that disruption of the IRS-1 gene does not affect insulin sensitivity or glucose metabolism in mice lacking the Kir6.2 gene (Figs. 2 and 3). These results indicate that KATP channel-associated increases in insulin action and glucose uptake are not mediated by the IRS-1 signal.
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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This study was supported by grants-in-aid from the Ministry of Education, Culture, Science, Sports, and Technology of Japan.
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FOOTNOTES
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K. Minami and M. Morita contributed equally to this work. Address for reprint requests and other correspondence: Susumu Seino, Division of Cellular and Molecular Medicine, Kobe Univ. Graduate School of Medicine, Kobe 650-0017, Japan (E-mail: seino{at}med.kobeu.ac.jp).
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
|
---|
- Aguilar-Bryan L, Clement JP IV, Gonzalez G, Kunjilwar K, Babenko A, and Bryan J. Toward understanding the assembly and structure of KATP channels. Physiol Rev 78: 227245, 1998.[Abstract/Free Full Text]
- Amoroso S, Schmid-Antomarchi H, Fosset M, and Lazdunski M. Glucose, sulfonylureas, and neurotransmitter release: role of ATP-sensitive K+ channels. Science 247: 852854, 1990.[ISI][Medline]
- Araki E, Lipes MA, Patti ME, Bruning JC, Haag B III, Johnson RS, and Kahn CR. Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372: 186190, 1994.[ISI][Medline]
- Ashcroft FM and Gribble FM. Correlating structure and function in ATP-sensitive K+ channels. Trends Neurosci 21: 288294, 1998.[ISI][Medline]
- Ashcroft FM, Harrison DE, and Ashcroft SJ. Glucose induces closure of single potassium channels in isolated rat pancreatic
-cells. Nature 312: 446448, 1984.[ISI][Medline]
- Bandyopadhyay G, Standaert ML, Sajan MP, Karnitz LM, Cong L, Quon MJ, and Farese RV. Dependence of insulin-stimulated glucose transporter 4 translocation on 3-phosphoinositide-dependent protein kinase-1 and its target threonine-410 in the activation loop of protein kinase C-
. Mol Endocrinol 13: 17661772, 1999.[Abstract/Free Full Text]
- Chutkow WA, Samuel V, Hansen PA, Pu J, Valdivia CR, Makielski JC, and Burant CF. Disruption of Sur2-containing KATP channels enhances insulin-stimulated glucose uptake in skeletal muscle. Proc Natl Acad Sci USA 98: 1176011764, 2001.[Abstract/Free Full Text]
- Clement JP IV, Kunjilwar K, Gonzalez G, Schwanstecher M, Panten U, Aguilar-Bryan L, and Bryan J. Association and stoichiometry of KATP channel subunits. Neuron 18: 827838, 1997.[ISI][Medline]
- Cook DL and Hales CN. Intracellular ATP directly blocks K+ channels in pancreatic B-cells. Nature 311: 271273, 1984.[ISI][Medline]
- Hajduch E, Alessi DR, Hemmings BA, and Hundal HS. Constitutive activation of protein kinase B alpha by membrane targeting promotes glucose and system A amino acid transport, protein synthesis, and inactivation of glycogen synthase kinase 3 in L6 muscle cells. Diabetes 47: 10061013, 1998.[Abstract]
- Ichikawa K, Akanuma Y, Kosaka K, and Kuzuya N. The effect of obesity on plasma triglycerides and fasting plasma IRI levels. Significance of insulinogenic index and glucose tolerance. Horm Metab Res 9: 429430, 1977.[ISI][Medline]
- Inagaki N, Gonoi T, Clement JP IV, Namba N, Inazawa J, Gonzalez G, Aguilar-Bryan L, Seino S, and Bryan J. Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270: 11661170, 1995.[Abstract]
- Inagaki N, Gonoi T, Clement JP, Wang CZ, Aguilar-Bryan L, Bryan J, and Seino S. A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron 16: 10111017, 1996.[ISI][Medline]
- Kohn AD, Kovacina KS, and Roth RA. Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase. EMBO J 14: 42884295, 1995.[Abstract]
- Kubota N, Tobe K, Terauchi Y, Eto K, Yamauchi T, Suzuki R, Tsubamoto Y, Komeda K, Nakano R, Miki H, Satoh S, Sekihara H, Sciacchitano S, Lesniak M, Aizawa S, Nagai R, Kimura S, Akanuma Y, Taylor SI, and Kadowaki T. Disruption of insulin receptor substrate 2 causes type 2 diabetes because of liver insulin resistance and lack of compensatory
-cell hyperplasia. Diabetes 49: 18801889, 2000.[Abstract]
- Kurth-Kraczek EJ, Hirshman MF, Goodyear LJ, and Winder WW. 5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle. Diabetes 48: 16671671, 1999.[Abstract]
- Miki T, Minami K, Zhang L, Morita M, Gonoi T, Shiuchi T, Minokoshi Y, Renaud J-M, and Seino S. ATP-sensitive potassium channels participate in glucose uptake in skeletal muscle and adipose tissue. Am J Physiol Endocrinol Metab 283: E1178E1184, 2002.[Abstract/Free Full Text]
- Miki T, Nagashima K, Tashiro F, Kotake K, Yoshitomi H, Tamamoto A, Gonoi T, Iwanaga T, Miyazaki J, and Seino S. Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice. Proc Natl Acad Sci USA 95: 1040210406, 1998.[Abstract/Free Full Text]
- Mu J, Brozinick JT Jr, Valladares O, Bucan M, and Birnbaum MJ. A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Mol Cell 7: 10851094, 2001.[ISI][Medline]
- Musi N, Fujii N, Hirshman MF, Ekberg I, Froberg S, Ljungqvist O, Thorell A, and Goodyear LJ. AMP-activated protein kinase (AMPK) is activated in muscle of subjects with type 2 diabetes during exercise. Diabetes 50: 921927, 2001.[Abstract/Free Full Text]
- Noma A. ATP-regulated K+ channels in cardiac muscle. Nature 305: 147148, 1983.[ISI][Medline]
- Ogawa W, Matozaki T, and Kasuga M. Role of binding proteins to IRS-1 in insulin signalling. Mol Cell Biochem 182: 1322, 1998.[ISI][Medline]
- Sakura H, Ammala C, Smith PA, Gribble FM, and Ashcroft FM. Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel subunit expressed in pancreatic
-cells, brain, heart and skeletal muscle. FEBS Lett 377: 338344, 1995.[ISI][Medline]
- Seino S. ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies. Annu Rev Physiol 61: 337362, 1999.[ISI][Medline]
- Spanswick D, Smith MA, Groppi VE, Logan SD, and Ashford ML. Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels. Nature 390: 521525, 1997.[ISI][Medline]
- Spruce AE, Standen NB, and Stanfield PR. Voltage-dependent ATP-sensitive potassium channels of skeletal muscle membrane. Nature 316: 736738, 1985.[ISI][Medline]
- Standaert ML, Bandyopadhyay G, Perez L, Price D, Galloway L, Poklepovic A, Sajan MP, Cenni V, Sirri A, Moscat J, Toker A, and Farese RV. Insulin activates protein kinases C-
and C-
by an autophosphorylation-dependent mechanism and stimulates their translocation to GLUT4 vesicles and other membrane fractions in rat adipocytes. J Biol Chem 274: 2530825316, 1999.[Abstract/Free Full Text]
- Standen NB, Quayle JM, Davies NW, Brayden JE, Huang Y, and Nelson MT. Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle. Science 245: 177180, 1989.[ISI][Medline]
- Sudo M, Minokoshi Y, and Shimazu T. Ventromedial hypothalamic stimulation enhances peripheral glucose uptake in anesthetized rats. Am J Physiol Endocrinol Metab 261: E298E303, 1991.[Abstract/Free Full Text]
- Tamemoto H, Kadowaki T, Tobe K, Yagi T, Sakura H, Hayakawa T, Terauchi Y, Ueki K, Kaburagi Y, Satoh S, Sekihara H, Yoshioka S, Horikoshi H, Furuta Y, Ikawa Y, Kasuga M, Yazaki Y, and Aizawa S. Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature 372: 182186, 1994.[ISI][Medline]
- Tanti JF, Gremeaux T, Grillo S, Calleja V, Klippel A, Williams LT, Van Obberghen E, and Le Marchand-Brustel Y. Overexpression of a constitutively active form of phosphatidylinositol 3-kinase is sufficient to promote Glut 4 translocation in adipocytes. J Biol Chem 271: 2522725232, 1996.[Abstract/Free Full Text]
- Terauchi Y, Iwamoto K, Tamemoto H, Komeda K, Ishii C, Kanazawa Y, Asanuma N, Aizawa T, Akanuma Y, Yasuda K, Kodama T, Tobe K, Yazaki Y, and Kadowaki T. Development of non-insulin-dependent diabetes mellitus in the double knockout mice with disruption of insulin receptor substrate-1 and
cell glucokinase genes. Genetic reconstitution of diabetes as a polygenic disease. J Clin Invest 99: 861866, 1997.[Abstract/Free Full Text]
- Tuttle RL, Gill NS, Pugh W, Lee JP, Koeberlein B, Furth EE, Polonsky KS, Naji A, and Birnbaum MJ. Regulation of pancreatic
-cell growth and survival by the serine/threonine protein kinase Akt1/PKB
. Nat Med 7: 11331137, 2001.[ISI][Medline]
- Whiteman EL, Cho H, and Birnbaum MJ. Role of Akt/protein kinase B in metabolism. Trends Endocrinol Metab 13: 444451, 2002.[ISI][Medline]
- Yamauchi T, Tobe K, Tamemoto H, Ueki K, Kaburagi Y, Yamamoto-Honda R, Takahashi Y, Yoshizawa F, Aizawa S, Akanuma Y, Sonenberg N, Yazaki Y, and Kadowaki T. Insulin signalling and insulin actions in the muscles and livers of insulin-resistant, insulin receptor substrate 1-deficient mice. Mol Cell Biol 16: 30743084, 1996.[Abstract]