Angiotensin II Enhances Insulin Sensitivity in Vitro and in Vivo

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Endocrinology 146(5):2246 –2254 Copyright © 2005 by The Endocrine Society doi: 10.1210/en.2004-1136

Angiotensin II Enhances Insulin Sensitivity in Vitro and in Vivo Chi-Chang Juan, Yueh Chien, Liang-Yi Wu, Wei-Ming Yang, Chih-Ling Chang, Ying-Hsiu Lai, Pei-Hsuan Ho, Ching Fai Kwok, and Low-Tone Ho Institutes of Physiology and Clinical Medicine, National Yang-Ming University, and Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan The renin-angiotensin system plays a critical role in the pathogenesis of obesity, obesity-associated hypertension, and insulin resistance. However, the biological actions of angiotensin II (AII) on insulin sensitivity remain controversial. Because angiotensinogen and AII receptors are expressed on adipose tissue, we investigated the effect of AII on the insulin sensitivity of isolated rat adipocytes. The results of a receptor binding assay showed the maximal AII binding capacity of adipocytes to be 8.3 ⴞ 0.9 fmol/7 ⴛ 106 cells and the dissociation constant to be 2.72 ⴞ 0.11 nM. Substantial expression of both type 1 and 2 AII (AT1 and AT2) receptors was detected by RT-PCR. AII had no effect on basal glucose uptake, but significantly potentiated insulin-stimulated glucose uptake; this effect was abolished by the AT1 antagonist, losartan. In addi-

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NGIOTENSIN II (AII) is the major effector hormone of the renin-angiotensin system (RAS), which regulates blood volume, arterial pressure, and cardiac and vascular function. AII binds to two distinct receptors, type I (AT1) and type II (AT2). AT1 receptors are widely distributed and mediate most of the biological responses that contribute to the known pressor effect of AII, whereas AT2 receptors antagonize several of the AT1 receptor-mediated responses; together, these two subtypes appear to coregulate the homeostasis of blood pressure and sodium excretion (1). RAS components were initially thought to exist only in the circulation, then were found in various tissues. After the liver, white adipose tissue is the most abundant source of angiotensinogen (2). The AII generated from adipose angiotensinogen elicits a variety of physiological effects when it binds to specific membrane receptors. Beside cardiovascular effects, adipose RAS has also been implicated in adipocyte growth and differentiation (3, 4). Moreover, overfeeding leads to increased local formation of angiotensinogen and AII from adipocytes in rats (5). Increased secretion of angiotensinogen from adipocytes may directly contribute to the First Published Online February 10, 2005 Abbreviations: AII, Angiotensin II; ⌬AUCglucose, change in the area under the glucose tolerance curve; BW, body weight; 2-DG, 2-deoxyglucose; GLUT4, glucose transporter 4; IR, insulin receptor; IR␤, insulin receptor ␤-subunit; IRS, insulin receptor substrate; KRB, Krebs-Ringer bicarbonate; OGTT, oral glucose tolerance test; pAkt, phospho-Akt; PI 3-kinase, phosphotidylinositol 3-kinase; RAS, renin-angiotensin system; SSPG, steady-state plasma glucose; SSPI, steady-state plasma insulin. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

tion, AII did not alter the insulin binding capacity of adipocytes, but increased insulin-stimulated tyrosine phosphorylation of the insulin receptor ␤-subunit, Akt phosphorylation, and translocation of glucose transporter 4 to the plasma membrane. AII potentiated insulin-stimulated glucose uptake through the AT1 receptor and by alteration of the intracellular signaling of insulin. Intraperitoneal injection of Sprague Dawley rats with AII increased insulin sensitivity in vivo. In conclusion, we have shown that AII enhances insulin sensitivity both in vitro and in vivo, suggesting that dysregulation of the insulin-sensitizing effect of AII may be involved in the development of insulin resistance. (Endocrinology 146: 2246 –2254, 2005)

close relationship between adipose tissue mass and blood pressure in mice (6). Furthermore, in human studies, local AII formation in adipose tissue is increased in obese hypertensive subjects (7, 8). Based upon these findings, the adipose RAS may play important roles in the pathogenesis of obesity, obesity-associated hypertension, and insulin resistance. However, the cellular mechanism of AII-induced metabolic disorders remains to be elucidated. Insulin acts on peripheral tissue to stimulate glucose metabolism or inhibit hepatic glucose output, and insulin sensitivity is the major determinant of insulin-dependent glucose utilization (9). Several vasoactive substances, such as norepinephrine (10), endothelin-1 (11), and nitric oxide (12), regulate insulin sensitivity and glucose uptake in insulin target tissues. These findings can also be extended in vivo and to some clinical studies (11, 13). Administration of an AT1specific antagonist ameliorates insulin resistance in fructosefed and Zucker fatty rats (14, 15). Blockade of the RAS also improves insulin sensitivity in patients with essential hypertension (16). However, administration of an AT1 antagonist has been reported to have no effect on insulin sensitivity in obese, nonhypertensive subjects with and without type 2 diabetes (17). Infusion of AII under euglycemic conditions increases insulin sensitivity in both healthy subjects (18, 19) and normotensive patients with noninsulin-dependent diabetes mellitus (20). Infusion of AII also increases glucose utilization during both sham and hyperinsulinemic glucose clamps (21). The role of AII in the regulation of insulin sensitivity has been widely investigated, but its effects are still controversial, and the underlying mechanisms remain unknown. In this study we showed that AII pretreatment leads to

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enhanced insulin-stimulated glucose transport in isolated rat adipocytes and that this effect is mediated by the enhancement of autophosphorylation of insulin receptor (IR) and the subsequent intracellular signaling. Furthermore, consistent with our in vitro findings, ip administration of AII also increased insulin sensitivity in rats. These findings demonstrated that AII enhances insulin sensitivity in vitro and in vivo. Materials and Methods Materials The anti-IR ␤-subunit (IR␤) and antiphosphotyrosine antibodies, horseradish peroxidase-conjugated secondary antibodies, and protein A-Sepharose were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-Akt and anti-phospho-Akt (pAkt) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiglucose transporter 4 (anti-GLUT4) antibodies were purchased from Chemicon International, Inc. (Temecula, CA). [3H]2-Deoxy-glucose, [125I]insulin, and [125I]AII were purchased from Amersham Biosciences (Aylesbury, UK). The Tri-Reagent Kit was purchased from Molecular Research Center, Inc. (Cincinnati, OH). All other chemicals were obtained from Sigma-Aldrich Corp. (St. Louis, MO).

Animals Male Sprague Dawley rats, weighing 400 –500 g, from the Animal Center of National Yang-Ming University were housed four to a cage in a temperature- and light-controlled room (20 –22 C; 12-h light, 12-h dark cycle; lights on at 0700 h) and were provided with regular diet chow and water ad libitum. The laboratory procedures used conformed to the guidelines of the Taiwan Government Guide for the Care and Use of Laboratory Animals. In the in vitro study, rats were killed for adipocyte preparation. In the in vivo study, 18 rats were injected ip with saline or AII [1 or 2 ␮g/100 g body weight (BW)], then, 30 min later, an oral glucose tolerance test (OGTT) was performed to evaluate insulin sensitivity.

Isolation of adipocytes After overnight fasting, the rats were killed by decapitation, and the epididymal fat pads from each group of rats (two or three animals) were pooled to isolate adipocytes using the Rodbell method (22) with minor modifications. Briefly, the fat tissue was minced and incubated for 1 h at 37 C in Krebs-Ringer bicarbonate (KRB) buffer containing 1% BSA and 0.1% collagenase in an oxygen-rich shaking chamber (CO2/O2, 5:95; 75 strokes/min). The suspension was then filtered through nylon mesh (400 ␮m pore size) and centrifuged at 100 ⫻ g for 1 min. The supernatant containing the adipocytes was harvested, and the cells were washed twice with, then resuspended in, KRB containing 1% BSA. The number of cells in the adipocyte suspension was determined after fixation with 2% osmium tetraoxide, and the lipocrit was measured before, during, and after each experiment to check cell viability.

AII binding to adipocytes Fifty microliters of [125I]AII (final concentration, 0.5 nmol/liter) and 50 ␮l KRB buffer containing increasing concentrations (1 pm to 1 ␮m) of unlabeled AII were mixed and added to 400-␮l aliquots of the adipocyte suspension (2 ⫻ 105 cells). The mixture was incubated for 60 min at 37 C in a 95% oxygen chamber with gentle shaking (75 strokes/min), then 300 ␮l of the suspension were transferred to a new centrifuge tube containing 200 ␮l silicon oil. The mixture was centrifuged at 1000 ⫻ g at room temperature for 1.5 min, then the cellular layer was transferred to a counting vial for measurement of radioactivity by a ␥-counter. A Scatchard plot was used to determine the number of AII binding sites and the dissociation constant.

RNA extraction Total RNA was extracted from treated rat adipocytes using a TriReagent Kit, its integrity was examined by 1% agarose gel electrophore-

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sis, and its concentration was determined by absorbance at 260 nm. All RNA samples were incubated with ribonuclease-free deoxyribonuclease I at 37 C for 30 min, followed by incubation at 100 C for 10 min to inactivate the deoxyribonuclease.

Determination of AT1 and AT2 receptor expression in adipocytes AT1 and AT2 receptor expression was detected by RT-PCR. Before RT, the RNA template was heated at 70 C for 5 min. RT was carried out at 42 C for 1 h in a total volume of 50 ␮l 1⫻ RT buffer, which contained 1 ␮g total RNA as a template, 5 U SUPER RT reverse transcriptase (HT Biotechnology Ltd., Cambridge, UK), 200 nm poly(deoxythymidine)12–18 primers (Promega Corp., Madison, WI), 200 mm of each deoxy-NTP (HT Biotechnology Ltd.), and 16 U human placental ribonuclease inhibitor (HT Biotechnology Ltd.). The RT mixtures were then heated at 100 C for 10 min to inactivate reverse transcriptase. For each PCR, the total volume of 50 ␮l 1⫻ buffer contained 5 ␮l RT template solution, 200 nm each of the sense and antisense primers, 1 U (0.2 ␮l) Pro Taq DNA polymerase (Protech Technology Enterprise Co. Ltd., Taipei, Taiwan), and 200 mm each of deoxy-NTP. The solution was overlaid with 30 ml mineral oil. PCR was performed in a DNA Thermal Cycler 480 (PerkinElmer, Norwalk, CT) with the following profile. After an initial denaturation at 94 C for 5 min, cycles of denaturation at 94 C for 30 sec, annealing at 55 C for 1 min, and elongation at 72 C for 1 min proceeded. In a preliminary run, we found that a minimum of 35 PCR cycles were required to produce an optimal amount of nucleic acids for measurement on an agarose gel. The last (35th) cycle was followed by a final extension step of 7 min at 72 C. The primers used (rat AT1 receptor sense primer, 5⬘-CCAGA AAAAC AAAAT GGCCC-3⬘; rat AT1 receptor antisense primer, 5⬘-TACAT TTCGG TGGAT GACAG-3⬘; rat AT2 receptor sense primer, 5⬘-AAGAG TGTAA GGATT GGGAG-3⬘; rat AT2 receptor antisense primer, 5⬘-TTCAG GGTCA GAAAA GAACC-3⬘) would amply fragments of 520 bp for AT1 receptor cDNA and 416 bp for AT2 receptor cDNA. Ten-microliter samples of each of the AII receptor PCR products amplified from the same RT template solution were electrophoresed on a 2% agarose gel, which was then stained with ethidium bromide.

Glucose uptake by adipocytes Basal and insulin-stimulated glucose uptake by adipocytes was determined by measuring 2-deoxyglucose (2-DG) transport into the cells, as described by Garvey et al. (23), with some modifications. To measure basal uptake, 400 ␮l fat cell suspension were preincubated with AII for various times at 37 C, then 50 ␮l [3H]2-DG (final concentration, 50 ␮m) were added, and incubation was continued for another 3 min. The reaction was terminated by adding 200 ␮l unlabeled 2-DG (final concentration, 0.14 m), then 300 ␮l of the suspension were transferred to a new vial containing 200 ␮l silicon oil and processed as described for the insulin binding assay. To measure insulin-stimulated glucose uptake, the cells preincubated with AII were mixed with 50 ␮l KRB buffer or increasing concentrations of insulin (final concentrations, 1 pm to 100 nm) 30 min before determination of glucose uptake.

Insulin binding to adipocytes Binding of insulin to adipocytes was measured as described previously (24). Briefly, 50 ␮l [125I]insulin (final concentration, 0.25 nmol/ liter) and 50 ␮l KRB buffer or increasing concentrations of unlabeled insulin (final concentrations, 1 pm to 1 ␮m) were mixed and added to 400-␮l aliquots of the adipocyte suspension (2 ⫻ 105 cells), the mixture was incubated for 30 min in a 95% oxygen chamber at 37 C with gentle shaking (75 strokes/min), and 300 ␮l of the suspension were transferred to a new centrifuge tube containing 200 ␮l silicon oil. The sample was then processed as described for the binding of AII.

Immunoprecipitation and immunoblotting After treatment, cells were lysed by sonication in cell lysis buffer (1% Nonidet P-40, 50 mm HEPES (pH 7.6), 250 mm NaCl, 10% glycerol, 1 mm EDTA, 20 mm ␤-glycerophosphate, 1 mm sodium orthovanadate, 1 mm sodium metabisulfite, 1 mm benzamidine hydrochloride, 10 ␮g/ml leupeptin, 20 ␮g/ml aprotinin, and 1 mm phenylmethylsulfonylfluoride).

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Immunoprecipitation was performed by incubating whole cell lysate (500 ␮g total protein) for 3– 4 h at 4 C with 5 ␮g anti-IR␤ antibody and 20 ␮l of a 50% suspension of protein A-Sepharose beads (25). The bead-bound immune complex was then washed five times in cell lysis buffer, and the proteins were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane for immunoblotting. The membrane was blocked by incubation for 30 min at room temperature with 5% skimmed milk in PBS, then incubated for 24 h at 4 C with antiphosphotyrosine, anti-IR␤, anti-Akt, or anti-pAkt antibody and for 30 min at room temperature with secondary antibody, followed by revelation with chemiluminescence reagent. To detect multiple signals from a single membrane, the membrane was treated for 20 min at 37 C with stripping buffer (59 mm Tris-HCl, 2% sodium dodecyl sulfate, and 0.75% 2-mercaptoethanol), then reblotted with a different antibody (26).

Subcellular fractionation Subcellular membranes were prepared as described by Leu et al. (27) with modification. Briefly, after treatment, the cells were washed twice with 10 ml buffer A (250 mm sucrose, 20 mm HEPES, 1 mm EDTA, and 1 mm phenylmethylsulfonylfluoride, pH 7.4). Cells were immediately homogenized with a homogenizer (PRO Scientific, Inc., Oxford, CT) in a 50-ml tube. The remainder of the homogenate was subjected to centrifugation at 16,000 ⫻ g at 4 C for 20 min. The pellet was resuspended in 5 ml buffer B (20 mm HEPES and 1 mm EDTA, pH 7.4) with sonicator, then applied to a 5-ml sucrose cushion (1.12 m sucrose in buffer B) and subjected to centrifugation at 100,000 ⫻ g at 4 C for 1 h. Plasma membranes removed from the top of the cushion were resuspended in buffer B and centrifuged at 30,000 ⫻ g for 30 min. Plasma membranes collected from the pellet were resuspended in buffer B to about 5 mg protein/ml. The supernatant from the 16,000 ⫻ g centrifugation was subjected to centrifugation at 250,000 ⫻ g for 1.5 h to collect the cytosolic microsomes, which were resuspended in buffer B to about 5 mg protein/ml.

Juan et al. • Angiotensin II and Insulin Sensitivity

Results Expression of AII receptor on rat adipocytes

Angiotensinogen and AII receptors are known to be expressed in adipose tissue (31). To characterize the AII receptors on isolated rat adipocytes, their AII binding capacity was measured by a competitive binding assay. Scatchard analysis of the results showed the maximal AII binding capacity of adipocytes to be 8.3 ⫾ 0.9 fmol/7 ⫻ 106cells and the dissociation constant to be 2.72 ⫾ 0.11 nm (Fig. 1A), demonstrating substantial expression of AII receptors on rat adipocytes. To determine the isoforms of the AII receptor expressed on adipocytes, we extracted total adipocyte RNA, performed RT-PCR using primers for AT1 and AT2 receptors, and detected substantial expression of both (Fig. 1B).

OGTT A 0-min blood sample was taken from each rat, then, without delay, the rats were given a glucose solution (0.2 g/0.1 ml/100 g BW) with gavage, and four additional blood samples were collected at 30, 60, 90, and 120 min. The concentration of plasma insulin was determined using an RIA technique developed in our own laboratory (28) with an antiporcine insulin antiserum, which cross-reacts 100% with human and rat insulin (29). Plasma glucose was measured on a glucose analyzer (model 23A, YSI, Inc., Yellow Springs, OH).

Steady-state plasma insulin (SSPI) and steady-state plasma glucose (SSPG) Measurements of SSPG and SSPI were performed as described previously (30). Briefly, on the day before the experiment, rats (two per condition) were anesthetized (sodium pentobarbital, 3 mg/100 g BW, ip), and the right jugular vein and left femoral veins were cannulated for blood-drawing and infusion, respectively. In the morning, a blood sample was drawn for reference, then somatostatin (0.1 mg/100 g BW䡠min) was infused for 30 min to suppress endogenous secretion of insulin. An additional blood sample was taken (zero time), and animals were injected with a bolus of AII (1 ␮g/100 g BW) or saline, then a mixed solution of 0.1 mg somatostatin, 0.27 mU insulin, and 0.8 mg glucose/100 g BW was infused per minute for a total period of 180 min. The volume of infused solution was 1 ml/h. Consequent blood samples were collected at 30, 60, 120, 135, 150, 165, and 180 min. PG and PI values were determined. The means of 135, 150, 165, and 180 min values were designated SSPI and SSPG.

Statistical analysis All results are expressed as the mean ⫾ sd. Differences between the two groups were analyzed by either Student’s t test or two-way ANOVA, with a post hoc t test when multiple measurements were made. Differences between the two groups were considered statistically significant at P ⬍ 0.05.

FIG. 1. AII binding capacity of and expression of AT1 and AT2 receptors on isolated rat adipocytes. A, Binding assays were performed as described in Materials and Methods; the inset shows the results of the Scatchard analysis. The values are the mean ⫾ SD for six experiments. B, Expression of AT1 and AT2 receptors detected by RT-PCR.

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Effect of AII on basal and insulin-stimulated glucose uptake

Although AII is reported to play a role in the regulation of insulin sensitivity (14 –21), its effects are diverse, and the underlying mechanism is not known. To elucidate the role of AII, we evaluated its effects on basal and insulin-stimulated glucose uptake by isolated adipocytes. As shown in Fig. 2, preincubation with AII had no effect on basal 2-DG uptake, and insulin treatment had the expected stimulatory effect (2.3-fold increase) on glucose uptake. However, in the presence of AII, insulin-stimulated glucose uptake was significantly increased, and the increase was dependent on the time of incubation with AII (Fig. 2). Fig. 3 shows the dose effect of insulin on glucose uptake and that AII potentiated insulin-stimulated glucose uptake at an insulin concentration of 10⫺11–10⫺7 m; the effect was significant at insulin concentrations of 10⫺10 m and higher. In the presence of AII, the plateau for insulin-stimulated glucose uptake increased significantly from 180 ⫾ 12 to 233 ⫾ 24 fmol/2 ⫻ 106 cells (P ⬍ 0.05). Together, these data show that AII augments insulin-stimulated glucose uptake response in isolated rat adipocytes. Involvement of AII receptor subtype on AII-enhanced insulin-stimulated glucose uptake

To examine which AII receptor isoform was involved in this effect, isolated adipocytes were preincubated with the AT1 receptor antagonist, losartan, or the AT2 antagonist, PD123319. As shown in Fig. 4A, the AT1 receptor antagonist abolished the effect of AII on insulin-stimulated glucose uptake, whereas the AT2 receptor antagonist had no effect (Fig. 4B). AII or losartan alone or AII plus losartan had no effect on basal glucose uptake. These data show that the effect of AII on insulin action was mediated by AT1 receptors. Effect of AII on IR binding

FIG. 3. AII promotes insulin-stimulated 2-DG uptake by adipocytes at various doses of insulin. Adipocytes were preincubated with (f; AII) or without (䡺; control) 10⫺7 M AII for 30 min, then for another 30 min with vehicle (B) or various concentrations (10⫺12–10⫺7 M) of insulin. Glucose uptake was determined as described in Materials and Methods. The values are the mean ⫾ SD for six experiments. *, P ⬍ 0.05 compared with the corresponding non-AII-treated control; #, P ⬍ 0.05 compared with insulin alone.

we examined whether insulin binding to adipocytes was altered by AII. When adipocytes were either untreated or pretreated for 1 h with 10⫺7 m AII, then tested in the competitive insulin binding assay, Scatchard analysis showed no difference in the insulin binding capacity or the dissociation constant (Fig. 5), showing that the effect of AII on the glucose uptake stimulatory response of insulin was not due to altered binding of insulin to isolated adipocytes. This suggests that the effect of AII in potentiating insulin action may result from alteration of postreceptor signaling of insulin.

To determine the mechanism involved in the action of AII on insulin-stimulated glucose uptake in isolated adipocytes,

FIG. 2. Time dependency of the effect of AII on glucose uptake in isolated adipocytes. Adipocytes were incubated with or without (䡺; control) 10⫺7 M AII for 1, 2, or 3 h, then with (insulin group) or without (basal group) 10⫺7 M insulin during the last 30 min of AII incubation. Thirty minutes after insulin addition, glucose uptake was determined as described in Materials and Methods. The values are the mean ⫾ SD for six experiments. *, P ⬍ 0.05 compared with the corresponding basal value.

FIG. 4. AII enhanced insulin-stimulated glucose uptake via the AT1 receptor on adipocytes. Adipocytes were incubated with 10⫺7 M AII and/or 10⫺6 M losartan (A) or 10⫺6 M PD123319 (B) for 1 h without insulin (basal group) or with 10⫺7 M insulin (insulin group). Insulin was added 30 min before the determination of [3H]-2 DG uptake. The values are the mean ⫾ SD for six experiments. *, P ⬍ 0.05 compared with insulin alone.

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FIG. 5. Lack of effect of AII pretreatment on the insulin binding capacity of adipocytes. Adipocytes were pretreated with 10⫺7 M AII (F; AII) or vehicle (E; control), then incubated with [125I]insulin plus various concentrations (10⫺10–10⫺6 M) of unlabeled insulin. Binding assays were performed as described in Materials and Methods. The values are the mean ⫾ SD for six experiments. B, Binding in the presence of test agents; Bo, maximal binding

Insulin receptor autophosphorylation, Akt phosphorylation, and GLUT4 translocation

To determine whether AII affected the autophosphorylation of the IR seen during insulin signaling, isolated adipocytes were incubated with 10⫺7 m AII and 10⫺7 m insulin for 10 min, then autophosphorylation of IR␤ was assessed by immunoprecipitation and Western blotting. As shown in Fig. 6, insulin stimulation resulted in IR␤ phosphorylation, and AII alone had no effect on IR␤ phosphorylation. However, cotreatment with AII plus insulin resulted in a significant enhancement of IR␤ phosphorylation compared with insulin alone. We also evaluated the phosphorylation of Akt, the kinase that mediates the downstream signaling of insulinstimulated glucose uptake. As shown in Fig. 7, the results indicated that Akt phosphorylation was stimulated by insulin and was further enhanced in AII-pretreated adipocytes. Because GLUT4 translocation is the key step before glu-

Juan et al. • Angiotensin II and Insulin Sensitivity

FIG. 7. Effect of AII on Akt phosphorylation. Adipocytes were incubated with 10⫺7 M AII and/or 10⫺7 M insulin or with medium alone for 10 min, then lysates were prepared. Phosphorylation of Akt was assessed by Western blotting with anti-pAkt and anti-Akt antibodies. The values are the mean ⫾ SD for three experiments. *, P ⬍ 0.05 compared with basal; #, P ⬍ 0.05 compared with insulin alone.

cose uptake, we examined whether AII affected the insulinstimulated translocation of GLUT4 by subcellular fractionation and Western blotting. As shown in Fig. 8A, AII alone exhibited no effect on GLUT4 content on plasma membrane compared with the basal level. Insulin treatment resulted in the expected increase in GLUT4 content in plasma membrane. Moreover, this stimulatory effect of insulin on plasma membrane GLUT4 content was further enhanced by AII pretreatment. Consistent with the findings on plasma membrane, the results indicated the concomitant decease in GLUT4 content in cytosolic microsomes of insulin-treated adipocytes (Fig. 8B). The GLUT4 content was further decreased in insulin-treated adipocytes by AII pretreatment. These findings demonstrated that AII pretreatment potentiated insulin-stimulated GLUT4 translocation from the cytosol to the plasma membrane, in agreement with the increase in glucose uptake. Changes in plasma glucose and insulin levels during OGTT

FIG. 6. Effect of AII on IR autophosphorylation. Adipocytes were incubated with 10⫺7 M AII and/or 10⫺7 M insulin or with medium alone for 10 min, then lysates were prepared. Autophosphorylation of IR␤ was assessed by immunoprecipitation with anti-IR␤ antibodies and Western blotting with anti-pTyr antibodies. The values are the mean ⫾ SD for three experiments. *, P ⬍ 0.05 compared with insulin alone.

The above findings demonstrated that AII potentiated insulin-stimulated glucose uptake and enhanced insulin sensitivity in isolated adipocytes. However, it remained unclear whether the insulin-sensitizing action of AII could be extended to the in vivo situation. To test the effect of AII in vivo, we performed an OGTT in male Sprague Dawley rats that had been acutely treated with either AII or normal saline. As shown in Fig. 9 and Table 1, in all three groups, baseline plasma glucose levels increased after oral glucose loading, and plasma insulin levels increased in response to oral glucose challenge. AII pretreatment caused a marked and dosedependent decrease in the change in the area under the glucose tolerance curve (⌬AUCglucose). In the group pretreated with 1 ␮g AII/100 g BW, the ⌬AUCinsulin (change in the area under the insulin profile curve during the OGTT) was similar to that in controls, indicating normal insulin secretion, whereas in the group pretreated with 2 ␮g AII/100 g BW, the ⌬AUCinsulin was lower, showing that insulin secretion was markedly suppressed, and the ⌬AUCglucose was

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FIG. 8. Effect of AII on GLUT4 translocation. Adipocytes were preincubated with or without 10⫺7 M AII for 30 min, then for another 30 min with or without 10⫺7 M insulin. The cells were homogenized, plasma membrane (A; PM) and cytosolic microsomal (B; CM) fractions were prepared, and the plasma GLUT4 content was analyzed by SDS-PAGE and immunoblotting with anti-GLUT-4 antibody. The values are the mean ⫾ SD for three experiments. *, P ⬍ 0.05 compared with basal; #, P ⬍ 0.05 compared with insulin alone.

also less than in the other two groups, indicating enhanced insulin sensitivity. These results demonstrate that AII also has insulin-sensitizing effects in vivo. Effects of AII on SSPI and SSPG

Additional evidence of the AII effect on insulin sensitivity in vivo is provided in Fig. 10. No significant difference in SSPI was found between control and AII-treated rats. However, the SSPG levels of AII-treated rats were significantly lower than those of control rats (73.73 ⫾ 5.747 vs. 117 ⫾ 9.355 mg/dl; P ⬍ 0.05). Consistent with our data from the OGTT, these results of SSPG and SSPI similarly demonstrated that AII-treated rats exhibited higher insulin sensitivity compared with control rats, indicating that AII enhances insulin sensitivity in vivo. Discussion

Insulin promotes glucose uptake in adipose tissue by activation of a series of signaling cascades. Much of this glucose is then converted to ␣-glycerophosphate, which is used in the esterification of fatty acids and permits their storage as triglycerides. To a minor extent, glucose can also be converted to fatty acids. Mice carrying an adipose tissue-specific dele-

tion of the GLUT4 gene rapidly develop marked muscular and hepatic insulin resistance (32), whereas mice carrying a muscle-specific deletion of GLUT4 develop hepatic and adipose insulin resistance secondary to the resulting hyperglycemia (33). These data suggest that adipose tissue plays an important role in whole body glucose homeostasis. Although adipose tissue glucose uptake accounts for only a small part of that for the whole body, the mechanism of insulin action in this tissue is of utmost importance. We used isolated adipocytes to explore the effect of AII on insulin sensitivity. AII potentiated insulin-stimulated glucose uptake in a time-dependent manner. Insulin alone stimulated glucose uptake in a dose-dependent manner, and the combination of AII plus various doses of insulin resulted in increased glucose uptake. The plateau value for insulin-stimulated glucose uptake was significantly enhanced, and the insulin binding capacity of the adipocyte was not changed by AII pretreatment. These findings suggested that the enhancing effect of AII on insulin action may be through alteration of intracellular signaling of insulin. Autophosphorylation of the IR␤ is the key step in insulinstimulated glucose uptake. As shown by the data in Fig. 6, the insulin-induced phosphorylation of this subunit was enhanced in isolated adipocytes pretreated with 10⫺7 m AII for 10 min. This enhanced phosphorylation of the IR would lead to hyperactivation of downstream molecules of insulin signaling, as the potentiating action of AII on insulin-induced Akt phosphorylation indicated. AII infusion has been shown to cause enhanced insulin signaling, including increased tyrosine kinase phosphorylation of the IR and IR substrates (IRSs), activation of phosphotidylinositol 3-kinase (PI 3kinase), and phosphorylation of Akt (34) in various insulin TABLE 1. Plasma glucose and plasma insulin parameters after OGTT

FIG. 9. Effects of AII on plasma glucose and insulin levels during the OGTT. Male Sprague Dawley rats were injected ip with AII (F, 1 ␮g/100 g BW; Œ, 2 ␮g/100 g BW) or normal saline (E); 30 min later, glucose (0.2 g/0.1 ml/100 g BW) was administered, and plasma levels of glucose (A) or insulin (B) were measured during the 120-min OGTT. The values are the mean ⫾ SD; n ⫽ 6. *, P ⬍ 0.05 compared with the saline group.

Treatment

Control (n ⫽ 6)

AII (1 ␮g/100 g BW) (n ⫽ 6)

AII (2 ␮g/100 g BW) (n ⫽ 6)

AUCglucose ⌬AUCglucose AUCinsulin ⌬AUCinsulin

284.5 ⫾ 5.8 82.8 ⫾ 5.4 57.5 ⫾ 3.9 18.9 ⫾ 2.7

269.1 ⫾ 11.0 54.4 ⫾ 6.4a 59.1 ⫾ 5.6 14.2 ⫾ 2.4

238.6 ⫾ 6.9a 35.8 ⫾ 7.1a 49.1 ⫾ 6.7 6.1 ⫾ 2.8a

Data are the mean ⫾ SD. Units: AUCglucose, mg/dl 䡠 h; AUCinsulin, ␮U/ml 䡠 h; insulin, ␮U/ml. a P ⬍ 0.05 compared with the control group.

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FIG. 10. Effects of AII on SSPI and SSPG levels. As described in Materials and Methods, rats were preinfused with somatostatin for 30 min. After AII or saline administration, rats were coinfused with the mixed solution of somatostatin, insulin, and glucose for 180 min. The mean plasma insulin and glucose levels during 135- to 180-min period were designated SSPI and SSPG. The values are the mean ⫾ SD; n ⫽ 4. *, P ⬍ 0.05 compared with the saline group.

target tissues. GLUT4 translocation is the key event in insulin-stimulated glucose uptake. In the present study, enhanced GLUT4 translocation was observed in adipocytes treated with AII plus insulin, in agreement with the observed AII-induced enhancement of insulin-stimulated glucose uptake. AII elicits the transactivation of tyrosine kinases for some receptors, such as IGF-I (35–37) and platelet-derived growth factor (38), and the subsequent phosphorylation of downstream signaling molecules, including IRSs (36, 37) and PI 3-kinase (35). In contrast to the AII-induced enhancement of insulin signaling, it has been reported that an AT1 receptor antagonist increases the insulin-induced phosphorylation of IRS-1, the association of IRS-1 with the p85 regulatory subunit of PI 3-kinase, PI 3-kinase activity, and GLUT4 translocation in skeletal muscles of diabetic mice (39). Likewise, insulin-induced Akt activation is inhibited by AII in the vasculature (40). Some in vitro studies in vascular smooth muscle cells also demonstrated that AII negatively modulates insulin signaling by stimulating multiple serine phosphorylation events in the early components of the insulin signaling cascade (41, 42). The influence of AII on insulin signaling remains controversial and seems to be tissue specific. Because AII and the RAS in adipose tissue are highly significant in whole body metabolism (2– 8), we believe that the AII-induced enhancement of insulin-stimulated signaling and glucose uptake in isolated adipocytes, demonstrated in this study, may play an important role in metabolism. However, the precise mechanism by which AII causes increased insulin-stimulated autophosphorylation of the IR remains to be determined. Hemodynamic factors have been suggested to be associated with glucose utilization. Vasodilator therapy is associated with enhanced insulin sensitivity (43), whereas infusion with the vasoconstrictor, norepinephrine, reduces forearm blood flow and results in decreased glucose utilization (44). To evaluate the effects of AII on insulin sensitivity in vivo, we analyzed the effects of ip injection of AII on insulin sensitivity in rats using the OGTT. AII pretreatment at doses of 1 and 2 ␮g/100 g BW did not alter blood pressure, suggesting that ip administration of AII at such doses may avoid AII-in-

Juan et al. • Angiotensin II and Insulin Sensitivity

duced-hemodynamic interference with insulin sensitivity. In a 120-min OGTT, AII-pretreated rats showed a dose-dependent decrease in the ⌬AUCglucose, suggesting that AII also enhances insulin sensitivity in vivo. Consistently, our measurements of SSPI and SSPG in AII-treated rats also showed the identical effect of AII on insulin sensitivity in vivo. Several clinical studies have demonstrated that AII increases insulin sensitivity under euglycemic conditions in healthy subjects and in normotensive patients with noninsulin-dependent diabetes mellitus (18 –20). However, AII infusion has also been suggested to result in insulin resistance in rats (45, 46). This discrepancy in insulin sensitivity between earlier findings and those of the present study may be attributable to differences in the experimental conditions, e.g. differences in the route of AII administration and the conscious vs. anesthetized status. It was reported that after ip injection of AII in rats, the plasma AII concentration immediately increased, reached a plateau at 15 min, gradually declined at 30 min, and was undetectable at 60 min (47). In our in vivo experiment, the doses of AII we applied were 1 and 2 ␮g/100 g BW, and the estimated elevation of plasma AII levels could be about 50 – 100 pg/ml. However, due to the short half-life of AII, to accurately detect the AII levels around fat cells is difficult. Numerous observations supported our findings (18 –20); the acute administration of AII should possess an insulinsensitizing property and enhance whole body insulin sensitivity. This outcome was determined by the summation of insulin-mediated glucose uptake in various insulin target tissues (e.g. skeletal muscle, liver, and adipose tissue) after AII administration. However, because adipose tissue serves a pivotal role in whole body glucose homeostasis and insulin sensitivity, the contribution of AII-enhanced glucose uptake in adipocytes should not be ignored, and it also may be of high physiological significance in glucose homeostasis. One issue that remains unclear is the precise mechanism of AII-mediated insulin resistance. In this study acute administration of AII potentiated insulin sensitivity in vitro and in vivo. Chronic infusion of AII has also been reported to cause hyperactivation of insulin signaling in several insulin target tissues, but insulin resistance developed in this study (34). Because the role of AII in white adipose tissue may be more important than its role in other tissues, we hypothesized that the potentiating action of AII on insulin-stimulated glucose uptake may play a significant role in normal and/or pathological states, such as obesity and obesity-related insulin resistance. Because insulin-stimulated glucose uptake in white adipose tissue is a key step in adipogenesis, the enhancing action of AII on insulin-stimulated glucose uptake may promote increased adiposity. Increased adiposity is highly associated with several metabolic problems, and the mechanisms involved have been suggested to be increased levels of free fatty acids; up-regulation of resistin, TNF-␣, and leptin; or down-regulation of adiponectin (48). Dysregulation of the action of AII may lead to increased adiposity (3, 49) and a variety of comorbidities, including insulin resistance. It is clear that the RAS is involved in many cardiovascular and metabolic diseases (50). AII in adipocytes is highly associated with adipose mass (6), and local formation of AII is

Juan et al. • Angiotensin II and Insulin Sensitivity

increased in obese hypertensive subjects (7, 8). Blockade of the RAS has been shown to increase insulin sensitivity and adiponectin concentrations in patients with essential hypertension (16). These results partially support our hypothesis of the role of AII in white adipose tissues and in possible obesity-related comorbidities. Another possibility is that AIImediated insulin resistance may be due to the abnormal hypersecretion of angiotensinogen and AII and the impairment of insulin signaling in adipose tissue. Chronic infusion of AII is also reported to induce enhanced insulin signaling, but signaling downstream of PI 3-kinase and Akt is impaired, and insulin-stimulated GLUT4 translocation from the cytosol to plasma membrane is reduced (34), whereas we observed increased insulin-stimulated GLUT4 translocation in response to AII. We presume that the decrease in insulinstimulated GLUT4 translocation in chronically AII-infused rats may be caused by desensitization of GLUT4 to insulin stimulation after long-term AII-induced hyperactivation of insulin signaling. In conclusion, we have shown that AII enhances insulin sensitivity both in vitro and in vivo and suggest that AIIinduced enhancement of insulin signaling in adipose tissue could be highly important. It is likely that insulin-stimulated glucose uptake is regulated by AII, at least on a short-term basis. Dysregulation of the action of AII on insulin-stimulated glucose uptake is expected to contribute to the pathologies of insulin resistance and several obesity-related metabolic disorders. Additional studies are required to fully understand the precise molecular mechanism of AII-induced enhancement of insulin signaling and the pathological changes in AII-induced insulin resistance. Acknowledgments Received August 26, 2004. Accepted January 25, 2005. Address all correspondence and requests for reprints to: Dr. LowTone Ho, Department of Medical Research and Education, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei, Taiwan. E-mail: [email protected] This work was supported by a research grant from the National Science Council of Taiwan (NSC 90-2314-B-075-029).

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