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J. Biol. Chem., Vol. 276, Issue 37, 34651-34658, September 14, 2001

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G_i2 Enhances in Vivo Activation of and Insulin Signaling to GLUT4^*

Xiaosong Song, Xilong Zheng, Craig C. Malbon, and Hsien-yu Wang^§¶

From the  Department of Molecular Pharmacology, University Medical Center, State University of New York, Stony Brook, New York 11794-8651 and the ^§ Department of Physiology and Biophysics, Diabetes and Metabolic Diseases Research Program, University Medical Center, State University of New York, Stony Brook, New York 11794-8661

Received for publication, June 25, 2001, and in revised form, July 10, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Heterotrimeric G-proteins, including G_i2, have been implicated in modulating glucose disposal and insulin signaling. This cross-talk between G-protein-coupled and tyrosine kinase-coupled signaling pathways is a focal point for the study of integration of cell signaling. Herein we study the role of G_i2 in modulating glucose transport, focusing upon linkages to insulin signaling. Utilizing mice harboring a transgene that directs the expression of a constitutively activated, GTPase-deficient mutant of G_i2 (Q205L) in adipose tissue, skeletal muscle, and liver, we demonstrate that G_i2 regulates the translocation of the insulin-sensitive GLUT4 glucose transporter in skeletal muscle and adipose tissue. The expression of Q205L G_i2 increased glucose transport and translocation of GLUT4 to the plasma membrane in vivo in the absence of insulin stimulation. Adipocytes from the Q205L G_i2 mice displayed enhanced insulin-stimulated glucose transport and GLUT4 translocation to the plasma membrane to levels nearly twice that of those from littermate controls. Phosphatidylinositol 3-kinase and Akt activities were constitutively activated in tissues expressing the Q205L G_i2. Studies of adipocytes from wild-type mice displayed short term activation of phosphatidylinositol 3-kinase, Akt, and GLUT4 translocation in response to activation of G_i2 by lysophosphatidic acid, a response sensitive to pertussis toxin. These data provide an explanation for the marked glucose tolerance of the Q205L G_i2 mice and demonstrate a linkage between G_i2 and GLUT4 translocation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The regulation of glucose transport in mammalian cells largely reflects the ability to translocate the insulin-sensitive glucose transporter GLUT4 (1-3) from intracellular microsomal sites to the cell membrane, where it facilitates glucose transport (reviewed in Refs. 4-7). Thus, understanding the intracellular trafficking of GLUT4 transporters has emerged as a critical element to our overall understanding of basal glucose disposal as well as insulin-stimulated glucose utilization. Important signal linkage maps have been created that implicate elements downstream from the insulin receptor in the insulin-sensitive translocation of GLUT4 (6, 8). These signal linkage maps include the phosphatidylinositol (PI)¹ 3-kinase (9) and several serine, threonine-specific protein kinases, including Akt (10-13), in the regulation of GLUT4. Earlier we reported a unique role for heterotrimeric G-protein G_i2 in modulating insulin signaling and glucose disposal (14-16). Deficiency of G_i2 in liver, adipose tissue, and skeletal muscle results in frank insulin resistance (16). Expression of a constitutively active form of G_i2, in sharp contrast, leads to enhanced glucose tolerance (17).

In the current work we sought to determine if activation of G_i2 signals to and thereby modulates glucose transport and GLUT4 translocation. We make use of the inducible, tissue-specific expression of the Q205L mutant form of G_i2 that is constitutively active in transgenic mice (iQ205L mice). When expressed in liver, adipose tissue, and skeletal muscle, Q205L G_i2 leads to increased glucose tolerance (17). This increase in glucose tolerance of iQ205L mice is observed even after treatment of the animals with the cytotoxic drug streptozotocin, rendering the mice insulin-deficient (18). Taken together, these data suggest the possibility that G_i2 may be regulating glucose transport activity in the absence of insulin. We explore this possibility and discovered increased glucose transport activity and GLUT4 abundance in the cell membrane of adipocytes from iQ205L mice. The adipose tissue of iQ205L mice display constitutively active PI 3-kinase activity, Akt activation, increased glucose transport activity, and a higher abundance of GLUT4 transporters in the cell membrane in the resting state. These results elucidate a new, important role of G_i2 in glucose disposal at the level of GLUT4.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Creating Transgenic Mice with Conditional, Tissue-specific Expression of Q205L G_i2-- Mice of the FVB strain were purchased from Taconic Farms (Germantown, NY). All animals were handled in accordance with the guidelines established by the Institutional Animal Care and Use Committee at SUNY/Stony Brook. Mice were maintained on a normal light/dark cycle. The transgenic mice were constructed using the rat Q205L G_i2 under the control of phosphoenolpyruvate carboxykinase promoter, as described elsewhere (17). The phosphoenolpyruvate carboxykinase promoter is silent in utero and is strongly activated at birth, yielding 2-3% of cellular mRNA in tissues targeted for expression, such as liver, skeletal muscle, and adipose tissue (19). Tail DNA samples were extracted, and the presence of Q205L G_i2 transgene was detected by polymerase chain reaction using primers described elsewhere (14). Targeted tissues such as epididymal white fat were taken to monitor the tissue-specific expression of Q205L G_i2 with anti-G_i2 antibody according to standard procedures (17). Six founder lines were bred for 10 generations, and mice used in these experiments were 8-16 weeks of age, with no age-related differences observed in the parameters measured.

Insulin Administration in Vivo-- Male transgenic and non-transgenic littermates or wild-type FVB male mice of the age were fed ad libitum and maintained on normal light/dark cycles. The evening before experiments, mice were allowed access to drinking water only. Experiments were routinely conducted between 8:00 and 10:00 a.m. Mice were anesthetized and insulin (5 IU) was administered via intravenous injection as described previously (20). Control mice received injections with vehicle alone.

Preparation of Cellular Fraction from Adipose Tissue and Skeletal Muscle-- Mouse epididymal fat pads or skeletal muscle from mouse hind limb were removed and trimmed off connective tissue. Then the fat or muscle tissue were minced and homogenized on ice in TES buffer containing 50 mM Tris-HCl, pH 7.0, 1 mM EDTA, pH 8.0, 0.1% -mercaptoethanol, 0.25 M sucrose, protease inhibitors (5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride), and 2.5 µM Na₃VO₄ using either a Polytron homogenizer for skeletal muscle or a Dounce glass/Teflon homogenizer for adipose tissue. The homogenate was subjected to centrifugation at 750 × g for 10 min. The pellet, which contained connective tissue and nuclei, was discarded. The supernatant was centrifuged at 16,000 × g for 30 min. The 16,000 × g pellet represented a plasma membrane-enriched fraction (PM). The supernatant was centrifuged at 48,000 × g for 20 min. The 48,000 × g pellet was enriched in high density microsomes (HDM). The supernatant was centrifuged at 200,000 × g for 60 min. The 200,000 × g pellet was enriched in low density microsomes (LDM), and the supernatant is referred to as the high speed supernatant (HS) fraction. All pellets were resuspended in TES buffer with standard protease inhibitors listed above. All centrifugation and subsequent steps were performed at 4 °C.

Protein and GLUT4 Distribution-- Mice (9-14 weeks in age), fasted over night before experiments, were anesthetized using a mixture containing ketamine (120 mg/kg body weight) and xylozine (8 mg/kg body weight), injected intraperitoneally. Once deep anesthesia was achieved, the abdominal cavity was opened and insulin (5 IU) or vehicle was administered via injection into inferior vena cava. At the time indicated after injection, epididymal fat pads and skeletal muscle from hind limb were removed. Protein determination was performed as described (21). Aliquots (100 µg of protein) from each subcellular fraction were subjected to SDS-polyacrylamide gel electrophoresis (PAGE). The resolved proteins were transferred electrophoretically to nitrocellulose blots and probed with anti-GLUT4 antibodies (Chemicon International, Temecula, CA). Upon secondary staining, the GLUT4 immune complexes were quantified on the blots by scanning densitometry. The total protein content was determined as was the relative abundance of GLUT4 per unit protein, providing an index of total and subcellular distribution of GLUT4. Throughout the manuscript the amounts of GLUT4 in the various subcellular fractions are reported as percents of the GLUT4 content.

Mouse Adipocyte Isolation and Subcellular Fractionation-- Adipocytes were isolated by collagenase digestion of mouse epididymal fat pads in glucose-free Krebs-Ringer phosphate buffer (12.5 mM HEPES, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO₄, 1 mM CaCl₂, 0.6 mM Na₂HPO₄, 0.4 mM NaH₂PO₄, pH 7.4) containing 4% bovine serum albumin at 37 °C for 30 min, as described (22). The digested tissue was washed 3 times in Krebs-Ringer phosphate with 2% bovine serum albumin. For insulin treatment, the isolated adipocytes were dispersed into two equal aliquots for cells from both the wild-type and transgenic mice and then pre-equilibrated at 37 °C for 30 min. Glucose (2.5 mM) with or without insulin (100 nM) was added to each tube, and the adipocytes were harvested after a 5-min incubation. Cells were homogenized by glass homogenizer for 10 strokes in HME buffer containing 20 mM HEPES, pH 7.0, 2 mM MgCl₂, 1 mM EDTA, and protease inhibitors (5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride). The homogenates were subjected to centrifugation at 500 × g for 5 min, and supernatant (post-nucleus fraction) was centrifuged at 16,000 × g for 30 min. This 16,000 × g pellet is a PM fraction.

Measurement of Glucose Transport-- Glucose transport activity was determined by analysis of the initial rates for the transport of 3-O-[³H]methylglucose in white fat cells acutely isolated from the iQ205L mice, as described earlier (22).

PI 3-Kinase Activity Assay-- PM or lysate fractions were resuspended in a reaction buffer containing 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM EGTA. An aliquot of membrane protein (5 µg) or lysate (100 µg) was added to the reaction buffer containing 20 mM MnCl₂, 0.1 mM phosphoinositol, phosphoserine (0.2 µg/µl), and 20 mM ATP (containing 1 µCi of [-³²P]ATP). The reaction was initiated by addition of the protein sample and maintained at 30 °C for 20 min. The reaction was stopped with 2 N HCl, and then an aliquot (160 µl) of chloroform:methanol (1:1) mixture was added. The samples were subjected to centrifugation at 10,000 × g for 1 min, and the lower phase was transferred to a new tube and washed by chloroform, methanol, 0.1 N HCl (1:1:1) twice. The lower phase was collected and dried under vacuum. The samples were resuspended in 10 µl of chloroform:methanol (95:5) then spotted onto the origin of a thin layer chromatography plate. The plate was developed in chloroform, methanol, NH₄OH (25%), H₂O (100:70:25:15). The resolved plate was dried and exposed to Kodak film or phosphoimaging cassette.

Akt Activity Assay-- The assay of Akt activity was performed using a kinase assay kit (New England Biolabs). Epididymal fat pads were homogenized in lysis buffer without Triton X-100 and subjected to centrifugation at 500 × g for 5 min. The supernatant was collected, and 1% Triton X-100 was added. Skeletal muscle from hind limb also was homogenized in lysis buffer and the slurry was rotated at 4 °C for 15 min. The samples were subjected to centrifugation at 20,000 × g for 30 min, and the supernatant was collected for immunoprecipitation. Aliquots of supernatant (100 µg of protein from adipose tissue or 200 µg of protein from skeletal muscle) was mixed with 20 µl of beads to which Akt antibody was immobilized, and the mixture was then rotated at 4 °C for 3 h in 500 µl of lysis buffer. The beads were washed three times, and the assay of Akt activity was performed using a glycogen synthase kinase (GSK)-3 fusion protein (M_r 30,000 kDa), as described by the vendor (Cell Signaling Technology, Beverly, MA). Akt activity was analyzed by immunoblotting. The blots were stained with phospho-GSK-3/(Ser21/9) antibody.

Treatment of Adipocytes with Pertussis Toxin and Lysophosphatidic Acid-- Pertussis toxin at 20 ng/ml was added to the collagenase digestion medium at the start of the adipocyte preparation. After washing of the isolated cells, 50 ng/ml pertussis toxin was added, providing a total of 4 h of intoxication. Cells were washed and then treated either with or without lysophosphatidic acid (LPA, 1 µM) for 15 min. Cells were collected and homogenized in HME buffer supplemented with a mixture of protease inhibitors (5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride) and 2.5 mM Na₃VO₄.

Gel Electrophoresis and Immunoblotting-- Protein samples were subjected to SDS-PAGE, transferred to a nitrocellulose blots, and stained with primary antibodies to the indicated antigens. The quantification of relative abundance of the immune complexes was performed by scanning densitometry.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transgenic mice carrying the pPEPCK expression vector harboring the GTPase-deficient mutant (Q205L) of G_i2 were newly created, characterized, and propagated (17, 23). The phosphoenolpyruvate carboxykinase promoter-based transgene directs robust expression of the Q205L G_i2 in adipose tissue, skeletal muscle, and liver, providing the mice with markedly enhanced glucose tolerance and insulin sensitivity (17). A possible linkage between G_i2 and insulin action was explored by testing the hypothesis that G_i2 regulates glucose transport. Mice with tissue-specific ablation of G_i2 display poor glucose tolerance (16), although no direct linkage between the heterotrimeric G-protein G_i2 and glucose transport has been established.

To test the hypothesis, we created four new founder lines, each carrying a pPEPCK Q205LG_i2 transgene that is expressed in target (adipose tissue, liver, and skeletal muscle), but not non-target (lung) tissues. Expression of the constitutively active Q205L G_i2 was found to be approximately equivalent to the amount of endogenous G_i2 alone in tissues from the control mice, thus effectively doubling the amount of immunoreactive G_i2 in the tissues from the iQ205L mice (Fig. 1). To explore if changes in glucose transport could explain some of insulinomimetic effects of expression of Q205L G_i2, hexose transport rates were measured in adipocytes of iQ205L mice and their non-transgenic littermates (Fig. 2). Initial rates of 3-O-[³H]methylglucose were determined in acutely prepared adipocytes. Adipocytes prepared from wild-type controls displayed a 4-5-fold increase in initial rates in response to insulin stimulation. The basal, unstimulated rates of hexose transport were 3-4-fold greater in the adipocytes from the iQ205L mice than in the wild-type controls. Insulin was able to stimulate increased hexose transport in the iQ205L mouse adipocytes, achieving a level of transport nearly 10-fold greater than the basal rates of adipocytes from the non-transgenic mouse controls. By comparing the unstimulated, basal states of hexose transport in the Q205L and control mouse adipocytes, it is clear that without stimulation by insulin, the transgenic mice adipocytes transport as much glucose as the control mice adipocytes in the presence of insulin.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Targeted expression of Q205L Gi2 in transgenic mice. Tissues were excised from Q205L Gi2 transgenic mice and either non-transgenic littermate or wild-type (WT) mice matched by sex and age. Plasma membrane-enriched subcellular fractions were prepared, and aliquots (40 µg of protein) were subjected to SDS-PAGE. Immunoblots of the resolved proteins were probed with anti-Gi2 antibodies.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Expression of Q205L Gi2 in adipocytes leads to enhanced glucose transport activity. Adipocytes were isolated from Q205L Gi2 transgenic mice and their wild-type counterparts. Adipocytes were assayed acutely for glucose transport activity in the absence or presence of 100 nM insulin by measurement of the initial rates of hexose transport using [3H]3-O-methylglucose uptake. Each assay was performed in triplicate. The data presented are mean values from at least four separate experiments.

Translocation of GLUT4 to the cell membrane appears to be the dominate feature of insulin action in adipose tissue and skeletal muscle (6). Analysis of GLUT4 translocation was performed first with the adipose tissue and skeletal muscle (gastrocnemius), examining the abundance of GLUT4 in four subcellular fractions: PM, HDM, HS, and LDMs (Fig. 3). The LDM fractions display the greatest membrane-associated fraction of GLUT4 in both adipose tissue and skeletal muscle. Within minutes of administration of insulin to the vena cava, GLUT4 translocates to the PM fraction, increasing the cell membrane-localized GLUT4 by 2-3-fold. The results from the two tissues, constituting major sites of insulin-stimulated glucose uptake, were quite similar with respect to GLUT4 translocation.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   GLUT4 in adipose and skeletal muscle of mice; translocation in response to insulin. Wild-type mice (9-14 weeks old) fasted overnight before the experiment were anesthetized with xylozine and ketamine and injected with either 5 IU of insulin or vehicle alone via the inferior vena cava. Tissues were excised 5 min post-administration of insulin, and various subcellular fractions were prepared. Aliquots (100 µg protein) were subject to SDS-PAGE. The abundance of the GLUT4 transporters was determined by immunoblotting with anti-GLUT4 antibodies followed by densitometry. The relative amounts of GLUT4 in each subcellular fraction were calculated from the specific activity and the volume of subcellular fractions. Total GLUT4 was calculated from the sum of each fraction. The percentage of total GLUT4 measured in each subcellular fraction was calculated. The values shown are means values ± S.E. (n = 9).

We compared the localization of GLUT4 between tissues obtained from the iQ205L mice and their non-transgenic, wild-type controls (Fig. 4). The abundance of GLUT4 was not altered in the iQ205L mice. The amount of GLUT4 localized to the PM fraction prepared from either adipose tissue or skeletal muscle in the absence of insulin was significantly greater in the iQ205L mice than in the wild-type controls (Fig. 4A). The changes in GLUT4 translocation were quantified from a number of separate experiments for both adipose tissue and skeletal muscle (Fig. 4, B and C, respectively). Tissue samples from iQ205L as compared with wild-type mice display two major differences in GLUT4 localization in the absence of added insulin; they are 1) increased levels of GLUT4 in the PM fractions and 2) relatively similar abundances of GLUT4 in the LDM fraction of adipose tissue while lower abundances of GLUT4 in LDM of skeletal muscle. These data obtained by immunoblotting of subcellular fractions and staining with anti-GLUT4 antibodies are in good agreement with the results from the measurements of hexose transport measured independently by analysis of 3-O-methylglucose uptake in adipocytes derived from the iQ205L and wild-type mice (Fig. 2). These studies provide a compelling case that expression of the constitutively active G_i2 results in greater abundance of GLUT4 in the cell membrane and that this distribution of GLUT4 contributes to the enhanced glucose tolerance of the iQ205L mice.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Q205L Gi2 enhances GLUT4 localization to plasma membrane in the absence of insulin. iQ205L mice or their wild-type (WT) counterparts (9-14 weeks of age) were fasted overnight before the experiment. After being anesthetized by ketamine and xylozine, mice were injected with either vehicle or 5 IU of insulin (INS) via the inferior vena cava. Tissues were excised, and subcellular fractions were prepared. Aliquots (100 µg of protein) were subjected to SDS-PAGE. A, GLUT4 distribution at resting stage (administration of vehicle only) in adipose tissue and skeletal muscle obtained from wild type and Q205L Gi2 transgenic mice. B and C, percentage of total GLUT4 measured in subcellular fractions prepared from mice after stimulation with or without insulin. The values shown are mean values ± S.E. (n = 9).

We explored the observation obtained in vivo by analysis of GLUT4 translocation in adipocytes prepared acutely from iQ205L mice and their controls (Fig. 5). The insulin response of GLUT4 translocation in vivo peaks at 5 min after administration of insulin to the vena cava (not shown). The GLUT4 translocation to the PM of adipocytes in response to insulin was observed to peak also at 5 min post-stimulation with insulin and was sustained for the next 30 min (Fig. 5A). Adipocytes from the iQ205L and control mice displayed the same time course (not shown). When measured in the absence of insulin, the abundance of GLUT4 in the PM was significantly greater in the adipocytes from the iQ205L mice than in their control counterparts (Fig. 5B), confirming the analysis of adipose tissue and skeletal muscle sampled in vivo (Fig. 3).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   GLUT4 translocation in adipocytes isolated from Q205L Gi2 transgenic versus wild-type control mice; effects of insulin stimulation. Adipose tissue was excised, and isolated adipocytes were prepared from iQ205L and wild-type (WT) control mice. Adipocytes were collected, washed three times, and then separated into aliquots in Krebs-Ringer phosphate buffer without glucose. Cells were treated with (+) or without () insulin (100 nM) plus 2.5 mM glucose for 5-30 min. After treatment, cells were homogenized, and plasma membrane-enriched subcellular fractions were prepared. Aliquots (20 µg of protein) were subjected to SDS-PAGE and immunoblotting with anti-GLUT4 antibodies. The relative amounts of GLUT4 were determined by scanning densitometry. A, time course of plasma membrane-localization of GLUT4 in adipocytes from wild-type mice. B and C, plasma membrane-associated GLUT4 abundance of adipocytes isolated from Q205L Gi2 transgenic mice or their wild-type counterparts after treatment without and with insulin (100 nM). The values shown were mean values ± S.E. (n = 4).

Insulin stimulated GLUT4 translocation in adipocytes from both the wild-type and iQ205L mice. Quantification of the amounts of PM-localized GLUT4 in adipocytes from the two sources reveals a 2-fold increase in the amount of GLUT4 for iQ205L mice adipocytes in the absence of insulin, remarkably a level of GLUT4 localization equivalent to wild-type mouse adipocytes in the presence of insulin (Fig. 5C). Thus, analysis of GLUT4 localization sampled in vivo as well as GLUT4 translocation and hexose transport sampled in isolated adipocytes in vitro provide compelling evidence for G_i2 regulating GLUT4 localization to the cell membrane in the absence of insulin stimulation. The fact that adipocytes of iQ205L mice with elevated hexose transport and GLUT4 abundance in the cell membrane still respond to insulin with increased GLUT4 translocation and hexose transport activity suggests the existence of an additional G_i2-mediated pathway for modulating GLUT4 translocation.

We explored signaling at the level of PI 3-kinase activity to evaluate a possible role of this key element of insulin signaling to GLUT4 translocation by G_i2. PI 3-kinase activity was sampled in isolated adipocytes and skeletal muscle prepared from iQ205L mice and their wild-type counterparts (Fig. 6). The adipocytes were treated with insulin in vitro, whereas determinations in skeletal muscle were made from mice that were administered insulin in vivo. The results in both adipocytes and skeletal muscle in the absence of insulin were qualitatively the same, i.e. the expression of Q205L G_i2 leads to constitutive activation of PI 3-kinase (Fig. 6, A and B). Administration of insulin leads to increased PI 3-kinase activation in adipocytes and skeletal muscle from both the iQ205L mice and control counterparts. In agreement with the results obtained by analysis of hexose transport and GLUT4 translocation (Figs. 2 and 4), the activation of PI 3-kinase in the iQ205L mice in the absence of insulin treatment was greater than or equal to the levels of PI 3-kinase activation observed in the wild-type mice after administration of insulin (Fig. 6, A and B). Results from an additional assay of PI 3-kinase activation, i.e. membrane localization of the p110 subunit of PI 3-kinase (Fig. 6C), confirmed the data provided by enzymatic assay of PI 3-kinase. The amount of p110 PI 3-kinase subunit associated with crude cell membranes was found to be significantly greater in adipocytes from the iQ205L G_i2 mice than in their normal controls.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Q205L Gi2 expression in vivo increases PI-3 kinase activity; analysis in adipose tissue and skeletal muscle A and C, adipocytes were isolated from both wild-type (WT) and Q205L Gi2 transgenic mice and then treated with (+) or without () insulin (100 nM) for 5 min. Cells were collected, and plasma membrane-enriched subcellular fractions were prepared. PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate. B, mice were deeply anesthetized by ketamine and xylozine and injected with either 5 IU of insulin or vehicle alone via the inferior vena cava. Skeletal muscle was excised, and crude plasma membrane-enriched fractions were prepared. For PI 3-kinase activity measurements, either 5 µg of protein of adipocyte subcellular fraction (A) or 50 µg of protein of skeletal muscle subcellular fraction (B) were assayed. Results obtained after thin layer chromatography of the reaction products were quantified by use of phosphoroimaging. C, analysis of PI 3-kinase p110 subunit abundance in plasma membrane-enriched subcellular fractions (20 µg of protein/lane) prepared from adipocytes. Samples were subjected to SDS-PAGE and immunoblotting. Immunoblots were stained with antibodies against the p110 PI 3-kinase subunit. The relative amounts of p110 subunit were determined by scanning densitometry. The values shown are means values ± S.E. (n = 4).

The phosphoinositide-dependent serine-threonine protein kinase Akt (also referred to as protein kinase B) has been proposed as an important link in the linkage from the insulin receptor, IRS1/2, and PI 3-kinase, to GLUT4 translocation and thereby to glucose uptake in muscle and fat (12). When measured in samples of adipose tissue from the iQ205L mice, activation of Akt in the absence of insulin administration was prominent, as determined using Ser-473 and Thr-308 phosphospecific antibodies to stain blots of Akt (Fig. 7). No changes were observed in the relative abundance of Akt by staining the blots with anti-Akt antibodies. Insulin stimulates increased activation of Akt in adipose tissue of both the iQ205L mice as well as the control mice. As noted above in measurements of hexose transport, GLUT4 abundance in the cell membrane and activation of PI 3-kinase, the activation of Akt in the iQ205L mice in the absence of insulin was greater than that of adipose tissue from control mice administered insulin in vivo. The activity of Akt was measured independently via measurement of the phosphorylation of a fusion protein harboring GSK 3, phosphorylation motifs and using, in tandem, phospho-specific antibodies that recognize both phospho-Ser-21 of GSK-3 and phospho-Ser-9 of GSK-3 (Fig. 7B). The immunoblotting and quantification results confirm that constitutive activation of Akt is associated with the expression of the Q205L G_i2 in adipose tissue of mice (Fig. 7B). The assay of Akt activity, in contrast to the immunoblotting data with phosphospecific-antibodies, suggests that Akt activity is already near maximal in the absence of insulin and that, when sampled from iQ205L mice administered insulin in vivo, no further increase in Akt activation was observed using this assay.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Increased phosphorylation and activity of Akt in adipose tissue of Q205L Gi2 transgenic mice. Mice (9-14 weeks old) were deeply anesthetized with ketamine and xylozine and then injected via the inferior vena cava with either 5 IU of insulin or vehicle alone for 5 min. A, adipose tissue was excised, and plasma membrane-enriched fractions were prepared. Aliquots (100 µg of protein) were subjected to SDS-PAGE and immunoblotting. The blots were probed with anti-phosphoserine 473 (S473-P) Akt antibodies, anti-phosphothreonine 308 (T308-P) Akt antibodies, or anti-Akt antibody. The relative abundance of each of the target proteins was determined by scanning densitometry. B, the 20,000 × g supernatant fraction of the lysate was collected. Lysate (0.1 mg of protein) was subjected to immunoprecipitation with beads to which anti-Akt antibodies were coupled. The beads then were washed three times, resuspended in 40 µl of reaction buffer supplemented with 1 µg of GSK-3 fusion protein (GSK-3K FP) and 200 mM ATP. The phosphorylation reaction was performed at 30 °C for 30 min. The supernatant was subjected to SDS-PAGE and immunoblotting. The blots were probed with phospho-specific antibodies that recognize phospho-Ser9 (GSK-3) or phospho-Ser-21 (GSK-3). The relative amounts of reaction products were determined by scanning densitometry. The values shown were means values ± S.E. (n = 5). WT, wild type.

When Akt phosphorylation and activation were sampled in skeletal muscle of iQ205L mice, a set of observations different from those in adipose tissue sampling were observed (Fig. 8). Phosphorylation of Ser-473 of skeletal muscle Akt displayed the same profile with regard to iQ205L versus control mice as well as with regard to without versus with insulin stimulation, as was noted in adipose tissue (Fig. 7). To our surprise, the phosphorylation of Thr-308 was largely unaffected by either the expression of the Q205L G_i2 or the stimulation with insulin in vivo. In four separate samplings, increased phosphorylation of Thr-308 was not detected. Levels of expressed Akt were similar to those observed in adipose tissue and relatively unchanged. To further explore the activation state of Akt in skeletal muscle, we employed the enzymatic assay (Fig. 8B). In good agreement with the immunoblotting data, the enzymatic assay failed to detect increased activity of Akt in skeletal muscle in response to expression of Q205L G_i2. Curiously, the enzymatic assay failed to detect activation of Akt in skeletal muscle of wild-type mice in response to insulin.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Akt phosphorylation in skeletal muscle of Q205L Gi2 transgenic mice is confined to Ser-473 and not Thr-308, rendering no activation. Mice (9-14 weeks old) were deeply anesthetized with ketamine and xylozine and then injected via the inferior vena cava with either 5 IU of insulin or vehicle alone for 5 min. A, tissues were excised, and plasma membrane-enriched subcellular fractions were prepared. Aliquots (100 µg of protein) were subjected to SDS-PAGE and immunoblotting. Blots were probed with anti-phosphoserine 473 (S473-P) Akt antibodies, anti-phosphothreonine 308 (T308-P) Akt antibodies, and anti-Akt antibodies. The relative abundance of the target proteins was determined by scanning densitometry. B, the 20,000 × g supernatant fraction of the lysate was collected. Lysate (0.1 mg of protein) was subjected to immunoprecipitation with beads to which anti-Akt antibodies were coupled. The beads then were washed three times, resuspended in 40 µl of reaction buffer supplemented with 1 µg of GSK-3 fusion protein (GSK-3K FP) and 200 mM ATP. The phosphorylation reaction was performed at 30 °C for 30 min. The supernatant was subjected to SDS-PAGE and immunoblotting. The blots were probed with phospho-specific antibodies that recognize phospho-Ser-9 (GSK-3) or phospho-Ser-21 (GSK-3). The relative amounts of reaction products were determined by scanning densitometry. The values shown were means values ± S.E. (n = 5).

The ability of Q205L G_i2 to influence GLUT4 translocation and activation of hexose transport illuminates an unexpected linkage between G_i2 and insulin action. The expression of a constitutively active version of G_i2 is invaluable in exploring new possible signal linkages between heterotrimeric G-proteins and tyrosine kinase actions. It is important, however, to be able to test if the results obtained in the iQ205L mice are demonstrable over the short term through receptor-mediated activation of G_i2. LPA is a potent stimulator of receptor-medicated activation of the G_i2 pathway. Adipocytes were isolated from wild-type mice and challenged with LPA, and the abundance of GLUT4 in the cell membrane-enriched fraction was assayed by immunoblotting (Fig. 9). LPA stimulated a marked translocation of GLUT4 to the cell membrane. Pertussis toxin, in contrast, is known to catalyze the ADP-ribosylation of G_i2, rendering it inactive. This LPA-stimulated translocation of GLUT4 was abolished by pretreated the adipocytes with pertussis toxin (Fig. 9A). The second read-out for these experiments was activation of PI 3-kinase. The products of PI 3-kinase activation were isolated by thin layer chromatography and abundance of inositol 3,4,5-trisphosphate was found to be increased dramatically in adipocytes challenged with LPA (Fig. 9B). This activation of PI 3-kinase was, like the GLUT4 translocation, abolished by pertussis toxin. Last, the activation and phosphorylation state of Akt was measured in adipocytes after acute stimulation with LPA (Fig. 9C). LPA increased by 50% the activity of Akt, measured using a GSK-3 fusion protein as a substrate. The phosphorylation of Ser-473 and Thr-308 also increased in response to LPA stimulation, albeit the phosphorylation of the Thr-308 residue was to a lesser extent. These data extend the observations derived from study of the iQ205L mouse and demonstrate a linkage between activation of G_i2 and GLUT4 translocation.

View larger version (28K): [in this window] [in a new window]   Fig. 9.   LPA stimulates GLUT4 translocation and activates PI-3 kinase and Akt in a pertussis toxin-sensitive manner. Mouse adipocytes were prepared from wild-type mice using tissue digestion buffer supplemented with 1 mg/ml collagenase in the presence (+) or absence of () 20 ng/ml pertussis toxin (PTX). After digestion, cells were washed free of collagenase and incubated with (+) or without () 50 ng/ml pertussis toxin to achieve a 4-h intoxication. Cells were treated with (+) or without () 1 mM lysophosphatidic acid for 15 min, collected, and used as a source of a post-nuclear, plasma membrane-enriched fraction. A and C, plasma membrane-enriched fractions (20 µg of protein) were subjected to SDS-PAGE, immunoblotting and staining with anti-GLUT4 antibodies. Aliquots of post-nuclear subcellular fraction (100 µg of protein) were employed for the assay of PI 3-kinase (B) and Akt activity (C), as described above. The relative abundance of the immunoblots (panels A and C) and of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) (panel B) was determined by scanning densitometry. S473-P, anti-phosphoserine 473; T308-P, anti-phosphothreonine 308.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Signaling via heterotrimeric G-protein-coupled pathways and tyrosine kinase pathways constitute the bulk of cell signaling (24). Elucidating how signals that originate from two or more of these pathways are integrated has evolved as a central task in modern biology. The convergence of G-protein-coupled signaling and tyrosine kinase action is best exemplified in the case of the well known counter-regulatory interactions between catecholamines and insulin. The 2-adrenergic receptor and insulin receptor converge in the control of metabolic regulation and include receptor-to-receptor cross-talk as well as multiple downstream overlaps that balance the demands of energy needs and availability of sources (25). The linkages between G-proteins such as G_q (26), G_i2 (16), and G_s (27) and insulin signaling have emerged in recent years. Mice deficient in G_i2 in target tissues like liver and adipose tissue display frank insulin resistance (16). Mice deficient in G_s display metabolic disturbances (28), whereas the G_s^/ mice are not viable (29). Heterozygotes for G_s(^/+) display enhanced insulin signaling (27). Understanding of the mechanisms by which deficiency in either G_i2 or G_s regulates insulin signaling is limited at best.

A complementary approach to the targeted knock-out of heterotrimeric G-proteins is the targeted overexpression of their  subunits or expression of GTPase-deficient, constitutively active forms in vivo. Complementing the targeted deficiency of G_i2 that provokes insulin resistance (16) is the creation of transgenic mice that express the constitutively active (Q205L) version of G_i2, which provokes enhanced glucose tolerance (17). In the current work we sought to understand one critical aspect of the iO205L mouse, its ability to tolerate and rapidly rectify a glucose load (17), even after insulin deficiency in response to streptozotocin (18). Examination of the hexose transport activity of adipocytes prepared from the iQ205L mice revealed increased initial rates of 3-O-methylglucose. The initial rates of hexose transport of adipocytes from iQ205L mice in the absence of insulin treatment were greater than those of the adipocytes from wild-type control mice in the presence of insulin. This same relationship was observed when the read-outs were extended to GLUT4 translocation to the cell membrane, to PI 3-kinase activation, and to Akt activation studied in adipocytes in vitro or in adipose tissue excised from mice treated with insulin in vivo. From these studies it can be concluded that expression of Q205L G_i2 does result in enhanced signaling from PI 3-kinase to enhanced abundance of GLUT4 in the cell membrane. Since GLUT4 translocation remains sensitive to insulin administration, the control by Q205L G_i2 does not appear to operate as precisely as insulin does, although clearly insulinomimetic in nature. These observations provide a wealth of understanding of why the iQ205L mice are so tolerant of glucose loading. Other G proteins have been implicated in modulating insulin action and GLUT4 translocation, especially G_q (26). These results were obtained from studies NIH 3T3-L1 cells in culture and add to the growing literature in which heterotrimeric G proteins and insulin signaling converge.

The details of the signal linkage path from expression of Q205L G_i2 and GLUT4 are not complete, but we provide some important features. The role of PI 3-kinase seems central, as PI 3-kinase activity has been reported to be essential for insulin signaling to GLUT4 (7, 30). We evaluated further the possible role of PI 3-kinase by the use of the inhibitor LY294002 to examine if inhibition of PI 3-kinase in adipocytes from iQ205L mice reverses the translocation of GLUT4, and it does not (not shown). In adipocytes, PI 3-kinase and Akt are activated constitutively by the expression of the Q205L mice, which according to the literature leads to GLUT4 translocation (10, 11). How Q205L G_i2 leads to the activation of PI 3-kinase remains unknown, although we have shown G_i2 to be linked to the activity of protein-tyrosine phosphatase (16). Further studies will be directed at providing these additional pieces to the puzzle.

Finally, it is important to address the limitation of studies targeting the expression of an activated G protein  subunit in vivo, namely adaptive changes. To address this concern we made use of LPA, a potent activator of G_i2, and pertussis toxin, which catalyzes the ADP-ribosylation and inactivation of G_i2. In acutely prepared mouse adipocytes, LPA stimulated activation of PI 3-kinase, Akt activation, and GLUT4 translocation. Each of these responses to LPA was abolished by prior treatment with pertussis toxin. Based upon the data gleaned from the iQ205L mice in vivo and their adipocytes in vitro, a compelling case can be made for G_i2 modulating GLUT4 as well as insulin signaling.

	FOOTNOTES

^* This work was supported by NIDDK, National Institutes of Health Grant DK30111 (United States Public Health Services) and by the American Cancer Society.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.

^¶ To whom correspondence should be addressed: Physiology and Biophysics, HSC, SUNY/Stony Brook, Stony Brook, NY 11794-8661. Tel.: 516-444-7873; Fax: 516-444-7696; E-mail: wangh@pharm.sunysb.edu.

Published, JBC Papers in Press, July 16, 2001, DOI 10.1074/jbc.M105894200

	ABBREVIATIONS

The abbreviations used are: PI, phosphatidylinositol; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; PM, a plasma membrane-enriched fraction; LDM, low density microsomes; HDM, high density microsomes; HS, high speed supernatant; PAGE, polyacrylamide gel electrophoresis; GSK, glycogen synthase kinase; LPA, lysophosphatidic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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