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Am J Physiol Endocrinol Metab 292: E779-E787, 2007. First published November 14, 2006; doi:10.1152/ajpendo.00407.2006
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Acute effects of wortmannin on insulin's hemodynamic and metabolic actions in vivo

Eloise A. Bradley, Michael G. Clark, and Stephen Rattigan

Biochemistry, Medical School, University of Tasmania, Hobart, Australia

Submitted 9 August 2006 ; accepted in final form 11 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, was systemically infused during a hyperinsulinemic euglycemic clamp to investigate its effects in vivo. Rats were infused under anesthesia with saline, 10 or 20 mU·min–1·kg–1 insulin, wortmannin (1 µg·min–1·kg–1) + saline, or wortmannin + insulin (10 mU·min–1·kg–1); wortmannin was present for 1 h before and throughout the 2-h clamp. Femoral blood flow (FBF), glucose infusion rate to maintain euglycemia (GIR), glucose appearance (Ra), glucose disappearance (Rd), capillary recruitment by 1-methylxanthine metabolism (MXD), hindleg glucose uptake (HLGU), liver, muscle, and aorta Akt phosphorylation (P-Akt/Akt), and plasma insulin concentrations were determined. Plasma insulin increased from 410 ± 49 to 1,680 ± 430 and 5,060 ± 230 pM with 10 and 20 mU·min–1·kg–1 insulin, respectively. Insulin (10 and 20 mU·min–1·kg–1) increased FBF, MXD, GIR, Rd, and HLGU as well as liver, muscle, and aorta P-Akt/Akt and decreased Ra (all P < 0.05). Wortmannin alone increased plasma insulin to 5,450 ± 770 pM and increased Ra, Rd, HLGU, and muscle P-Akt/Akt without effect on blood glucose, FBF, MXD liver, or aorta P-Akt/Akt. Wortmannin blocked FBF, MXD, and liver P-Akt/Akt increases from 10 mU·min–1·kg–1 insulin. Comparison of wortmannin + 10 mU·min–1·kg–1 insulin and 20 mU·min–1·kg–1 insulin alone (both at ~5,000 pM PI) showed that wortmannin fully blocked the changes in FBF and Ra and partly those of GIR, Ra, Rd, HLGU, and muscle P-AKT/Akt. In summary, wortmannin in vivo increases plasma insulin and fully inhibits insulin-mediated effects in liver and aorta and partially those of muscle, where the latter may result from inhibition of insulin-mediated increases in blood flow and capillary recruitment.

glucose uptake; blood flow; microvasculature; insulin action

WORTMANNIN IS A low-molecular-weight hydrophobic compound with a sterol-like structure (32) and was first demonstrated to inhibit cellular responses to receptor stimulation in 1987 (2, 12). In those experiments, submicromolar amounts of wortmannin were found to inhibit the respiratory burst of neutrophils triggered by a variety of receptor ligands, including N-formylmethionylleucylphenylalanine (fMLP), leukotriene B4, or platelet activating factor. However, it became clear that the inhibitory effects of wortmannin were specific to certain types of G protein-coupled receptors (30), and not processes triggered by the phorbol esters (32). In fat cells, micromolar amounts of wortmannin were shown to inhibit insulin-mediated glucose uptake (20). The target for wortmannin inhibition in these cells was identified to be at the level of phophatidylinositol 3,4,5-trisphosphate formation from phosphatidylinositol 4,5-bisphosphate by the enzyme phosphoinositide (PI) 3-kinase. The concentrations of wortmannin required for the inhibition of PI 3-kinase in neutrophils were similar to those reported for the inhibition of fMLP-stimulated superoxide production by the same cells. PI 3-kinase was common to both signaling cascades, one involving G protein-coupled fMLP receptors, and the other leading to superoxide formation (1, 12). For the fat cell insulin signaling systems, stimulated PI 3-kinase activity was detected in membrane fractions and in immunoprecipitates targeted for tyrosine-phosphorylated proteins. Wortmannin was found to block these increases in PI 3-kinase activity due to insulin and further downstream metabolic outcomes resulting from insulin action, including 2-deoxyglucose uptake (20), opposition of isoproterenol-stimulated lipolysis (20, 22), and cAMP phosphodiesterase activation (22). Wortmannin at nanomolar concentrations that inhibit isolated PI 3-kinase inhibits insulin-mediated glucose uptake in muscle (15, 39). In contrast, both hypoxia and muscle contraction that each act to increase glucose uptake are not susceptible to inhibition by wortmannin (39).

Wortmannin has proven to be membrane permeant and to potently inhibit (IC50 ~3 nM) PI 3-kinase in all mammalian tissues tested. It appears to bind irreversibly to the p110 catalytic subunit of PI 3-kinase (38), possibly involving the alkylation of a lysine residue at the ATP-binding site (37). Even at concentrations up to 1 µM wortmannin does not inhibit myosin light-chain kinase, protein kinases A, C, and G (38), or kinases downstream of PI 3-kinase in insulin signaling (9). However, there are some reports of wortmannin inhibition of phospholipases (e.g., phospholipase A2; see Ref. 10), and wortmannin displays little selectivity within the PI 3-kinase family (30); thus, a broad number of processes have the potential to be inhibited when exposure to wortmannin is for extended periods (30).

Wortmannin has been used in vivo by others to test effects on tumor growth (3, 11, 29) and bone resorption (26), but not to our knowledge to assess acute effects on insulin action. Our interest in using wortmannin was to test the hypothesis that insulin-mediated capillary recruitment in vivo is wortmannin sensitive. We have reported previously that insulin-mediated capillary recruitment is an early (33) and sensitive (43) action of insulin in muscle, and we have proposed that, by increasing microvascular perfusion, delivery of insulin and nutrients is enhanced. Unlike insulin actions within myocytes, which can be studied in vitro, capillary recruitment, which requires intact associations between the microvasculature and muscle, must be studied in vivo. Thus, in the present study, we applied wortmannin acutely before and during a hyperinsulinemic euglycemic clamp in rats to assess effects on hemodynamic and metabolic responses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Experimental protocols were approved by the University of Tasmania Animal Ethics Committee. Male hooded Wistar rats (University of Tasmania Animal House, Hobart, Tasmania, Australia) weighing 245 ± 3 g were raised on a commercial diet (Pivot, Launceston, Tasmania, Australia) containing 21.4% protein, 4.6% lipid, 68% carbohydrate, and 6% crude fiber with added vitamins and minerals together with water ad libitum. Until the day of surgery, rats were housed at a constant temperature of 21 ± 1°C with a 12:12-h light-dark cycle. Food and water were freely available until animals were administered anesthetic.

Experimental protocols. These are shown in Fig. 1 and were carried out in anesthetized rats as described previously (24). Anesthesia was necessary to prevent movement effects on capillary recruitment and was introduced using Nembutal (sodium pentobarbitone), 50 mg/kg body wt, and maintained for the duration of the experiment using a continual infusion of 0.6 mg·min–1·kg–1 via the left jugular cannula. Surgical preparation for the protocols involved cannulation (PE-50, Intramedic; Beckton-Dickinson, Parsippany, NJ) of the carotid artery for arterial sampling [blood glucose (BG)] and continuous measurement of heart rate (HR) and blood pressure (BP; pressure transducer Transpac IV; Abbott Critical Care Systems, Morgan Hill, CA) and both jugular veins for continuous administration of anesthetic and other intravenous infusions. In addition, a tracheotomy tube was inserted so that the animal could spontaneously breathe room air throughout the course of the experiment. A small incision (1.5 cm) was made in the skin overlaying the femoral vessels of the left leg, the femoral artery was separated from the femoral vein and saphenous nerve, and an ultrasonic flow probe (VB series 0.5 mm; Transonic Systems, Ithaca, NY) was positioned around the femoral artery just distal to the rectus abdominis muscle before the point where the epigastric artery leaves the femoral artery. After placement of all cannulas and the Transonic flow probe and following a 1-h equilibration, rats were infused for 3 h with either 0.5% (vol/vol) DMSO or wortmannin (1 µg·min–1·kg–1; Sigma Chemical, St. Louis, MO) in 0.5% DMSO as shown in Fig. 1. Wortmannin infusion was initiated by a bolus injection of 2.5 µg in 0.10 ml at time (t) = –60 min. One hour later (t = 0 min) saline or insulin (10 mU·min–1·kg–1, Humulin R; Eli Lilly, Indianapolis, IN) infusion was commenced (Fig. 1). Insulin infusions (20 mU·min–1·kg–1) were made only with vehicle (DMSO) treatment. In rats receiving insulin, BG was maintained at the initial level by the infusion of 30% (wt/vol) glucose; GIR represented the rate of glucose infusion at the time stipulated or the steady-state rate at the end of the experiment (t = 120 min). At the end of the experiment, blood was sampled from the femoral vein and carotid artery. From the arteriovenous difference multiplied by the flow, hindleg glucose uptake (HLGU) was calculated. A primed (74 kBq) continuous infusion (3.7 kBq/min) of [3-3H]glucose (Amersham Life Science, Castle Hill, NSW, Australia) was administered during the final 1 h. Arterial plasma samples taken at 75, 90, 105, and 120 min were deproteinated, evaporated to dryness to remove 3H2O, and resuspended, and [3H]glucose radioactivity was determined. The rate of appearance (Ra) and rate of disappearance (Rd) of glucose were calculated using the isotope dilution equation

Formula
where GIR is glucose infusion rate, F is the rate of tracer infusion, and SA is the specific activity of glucose (5). SA was calculated from the plasma radioactivity divided by the glucose concentration.


Figure 1
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  		Fig. 1. Experimental protocol. After surgical preparation for the hyperinsulinemic euglycemic clamp and a period of equilibration of ~60 min, 0.5% (vol/vol) DMSO or wortmannin dissolved in DMSO was infused starting with a bolus of 2.5 µg followed by constant infusion (jugular vein) for 3 h at 1 µg·min–1·kg–1. Saline or insulin infusions (10 or 20 mU·min–1·kg–1) were commenced 60 min later [time (t) = 0 min] and continued for 120 min. Infusion of 30% (wt/vol) glucose was commenced shortly after the commencement of the insulin infusion at a rate to maintain euglycemia as assessed from blood samples (drops). At 60 min, a bolus injection of allopurinol (10 µmol/kg) was made followed by infusions of 1-methylxanthine (1-MX) and [3-3H]glucose. Infusions are indicated by the bars. Arterial and femoral vein blood samples [arteriovenous (A-V)] were taken at the end of the experiment to determine hindleg glucose and 1-MX extraction. Liver, muscle, and aorta samples were also taken at this time to determine phosphorylated Akt (P-Akt) and Akt total. There were 5 experimental groups (n = 8/group) as follows: 0.5% DMSO for 3 h (vehicle for wortmannin) + saline for 2 h; DMSO + insulin (10 mU·min–1·kg–1); DMSO + insulin (20 mU·min–1·kg–1); 1 µg·min–1·kg–1 wortmannin + saline; wortmannin + insulin (10 mU·min–1·kg–1).

 
Plasma insulin was also determined on the final arterial blood sample by ELISA assay (Mercodia).

Femoral blood flow and capillary recruitment. Femoral blood flow (FBF) was continuously measured from a Transonic flow probe positioned around the femoral artery of one leg. Capillary recruitment was determined by measuring the metabolism of infused 1-methylxanthine (1-MX), a substrate targeted for xanthine oxidase, that in the hindleg muscle is located predominantly in capillary endothelium (16). 1-MX (Sigma Chemical) was infused at a constant rate (0.5 mg·min–1·kg–1; Fig. 1) to maintain a steady-state arterial level of ~20 µM. As noted previously (24, 25), this was achieved by partly inhibiting the activity of xanthine oxidase with allopurinol (10 µmol/kg; see Ref. 13) administered as a bolus dose 5 min before commencing the 1-MX infusion (Fig. 1). This not only allowed constant arterial concentrations of 1-MX to be maintained throughout the experiment, but lowered the Km of the enzyme sufficiently so that 20 µM 1-MX was well above saturation (unpublished observations).

Plasma (100 µl) from arterial and leg venous blood samples taken at the end of the experiment was mixed with 20 µl of 2 M perchloric acid and centrifuged for 10 min. The supernatant was used to determine 1-MX, allopurinol, and oxypurinol concentrations by reverse-phase HPLC as previously described (24). Capillary recruitment, expressed as 1-MX metabolism (MXD), was calculated from arteriovenous blood 1-MX concentration difference and multiplied by FBF at the end of the experiment (t = 120 min). The concentration of 1-MX in blood was calculated from the plasma concentration by taking into account the volume accessible to 1-MX. This was determined from plasma concentrations obtained after additions of standard 1-MX to whole rat blood, where it was found that blood 1-MX concentration was 87.1% of the plasma concentration. The rate of MXD across the hindleg (expressed as nmol/min) reflects conversion by capillary endothelial xanthine oxidase to the sole product, 1-methyl urate (23). This method has been found to correlate well with other techniques that measure capillary recruitment (6).

Akt phosphorylation. Liver, thoracic aorta, and lower leg muscles (principally the gastrocnemius group) were freeze-clamped (muscles and liver in situ; aorta as quickly as possible after removal) at the end of the experiment using liquid nitrogen-cooled tongs and stored at –80°C until assayed. The frozen tissue samples were ground into a fine powder under liquid nitrogen and homogenized in (1:60 wt/vol) ice-cold solubilizing buffer with a Silent Crusher S (Heidolph). The solubilization buffer was composed of 25 mM Tris (pH 7.5), 2.6 M potassium fluoride, and 250 mM EDTA. The insoluble material was removed by centrifugation at 13,000 rpm in a Biofuge pico (Heraeus) for 10 min at 4°C. The protein concentration of the supernatants was determined using Bio-Rad Protein Assay (Bio-Rad). Aliquots of the resulting supernatants containing 10 µg of protein were then heated with 70 mM Tris-buffered (pH 6.8) SDS (2%) for 5 min at 90°C. Heat-treated aliquots were then subjected to SDS gel electrophoresis. Electotransfer of proteins from the gel to nitrocellulose membranes was performed for 2 h at 30 volts (constant voltage) at 4°C. After transfer, the membranes were blocked and then incubated with the primary antibody phospho-Akt (Ser473) or Akt antibody (Cell Signaling) overnight at 4°C. Membranes were then washed and incubated with horseradish-linked antibody (Cell Signaling) diluted in blocking buffer for 1 h at room temperature. The membranes were developed using the enhanced chemiluminescence method (Progen Biosciences). Bound antibodies were detected by exposing to X-ray film (Hyper Film; Amersham Biosciences). Band intensities were quantified by optical density using DScan EX software version 3.1.

Statistics. One-way, two-way, or two-way repeated-measures ANOVA was performed using SigmaStat (SPSS Science, Chicago, IL), with comparisons made between conditions using the Student-Newman-Keuls post hoc test. Significance was assumed at the level of P < 0.05. Data are presented as means ± SE; if error bars are not visible, they are within the symbol.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Plasma insulin and glucose. Table 1 shows BG and plasma insulin values measured at the end of the experiment (t = 120 min) for the five protocols: vehicle (0.5% vol/vol DMSO) and saline, vehicle and insulin (10 mU·min–1·kg–1), vehicle and insulin (20 mU·min–1·kg–1), wortmannin and saline, and wortmannin and insulin (10 mU·min–1·kg–1). BG values were similar for all five protocols (i.e., ~5 mM). In three of the protocols involving insulin infusion, concentrated glucose solution was infused to maintain the value at euglycemia. The plasma insulin values showed marked differences. Insulin infusion increased the basal value of 410 ± 49 pM to 1,680 ± 430 and 5,060 ± 230 pM at 10 and 20 mU·min–1·kg–1, respectively. Wortmannin with saline also markedly increased plasma insulin to ~5,000 pM (Fig. 2), and this was not further increased by coinfusion of 10 mU·min–1·kg–1 insulin with wortmannin. Thus all three of vehicle and 20 mU·min–1·kg–1 insulin, wortmannin and saline, and wortmannin and 10 mU·min–1·kg–1 insulin had a plasma insulin value of ~5,000 pM at t = 120 min (Table 1).


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  		Table 1. Blood glucose and plasma insulin values at the end of the experiment

 

Figure 2
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  		Fig. 2. Time course for the effect of wortmannin on plasma insulin. Details are as given in Fig. 1. Values are means ± SE (n = 3). *Significantly different (P < 0.05) from t = –60 min values.

 
Hemodynamic changes. Figure 3 shows time courses for changes in HR, BP, FBF, and vascular resistance (VR) over the preceding 60 min before and during saline (control) or 120 min hyperinsulinemic euglycemic clamp. Relative to vehicle, wortmannin caused a time-dependent gradual decrease in HR over 60 min (P < 0.05) that reversed in the final 60 min to near basal position; the time course was similar whether or not insulin was present (Fig. 3, A and B). There was also an increase in BP that accompanied the fall in HR and which was significant after 45 min (t = –15 min; Fig. 3C). An increase in BP due to wortmannin also occurred in the presence of insulin (Fig. 3D).


Figure 3
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  		Fig. 3. Time course for the effect of wortmannin on changes in heart rate (HR), blood pressure (BP), femoral arterial blood flow (FBF), and femoral vascular resistance (VR) in control (saline) and insulin infusions. Details are as given in Fig. 1. Values are means ± SE for saline ({circ})-, wortmannin (bullet)-, insulin (10 mU·min–1·kg–1, {square})-, insulin (20 mU·min–1·kg–1, {blacksquare})-, and wortmannin + insulin (10 mU·min–1·kg–1, {blacktriangledown})-treated rats. *Significant difference between wortmannin + saline treatment and vehicle + saline treatment (A, C, E, and G). #Significant difference between wortmannin + insulin treatment and both vehicle + insulin treatments (B, D, F, and H).

 
Figure 3, E and F, shows the effects of wortmannin and insulin on changes in FBF. Basal FBF was 0.81 ± 0.15 ml/min, and this was increased by ~0.4 ml/min by 10 and 20 mU·min–1·kg–1 insulin. Wortmannin plus saline, as well as wortmannin plus 10 mU·min–1·kg–1 insulin each tended to decrease FBF to values less than basal that by the end of the experiment were significant (P < 0.05; Fig. 3, E and F). Thus wortmannin with 10 mU·min–1·kg–1 insulin blocked the stimulatory effect of this dose of insulin on FBF. Also, wortmannin with 10 mU·min–1·g–1 insulin produced an FBF that was significantly less than that of 20 mU·min–1·kg–1 insulin (Fig. 3F) even though the plasma insulin values were similar (Table 1).

Figure 3, G and H, shows the effects of wortmannin on changes in VR. Wortmannin caused a significant increase in VR that commenced from 30 min and continued to increase throughout the rest of the experiment.

Capillary recruitment, measured by MXD and determined from arteriovenous difference and FBF at the end of the experiment (t = 120 min), is shown in Fig. 4. Insulin (10 mU·min–1·kg–1) significantly increased MXD relative to control. Wortmannin alone was without effect but when present with insulin completely blocked the stimulation due to 10 mU·min–1·kg–1 insulin.


Figure 4
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  		Fig. 4. Effect of wortmannin, insulin, and a combination of wortmannin + insulin on hindleg muscle capillary recruitment determined from 1-MX metabolism. Details are as given in Fig. 1. Values were determined at the end of the experiments (t = 120 min) and are means ± SE for vehicle-treated rats with either saline or 10 mU·min–1·kg–1 insulin (open bars) and wortmannin-treated rats with either saline or 10 mU·min–1·kg–1 insulin (filled bars). *Insulin-treated significantly different (P < 0.05) from corresponding saline-treated values. #Wortmannin-treated significantly different from corresponding vehicle-treated values.

 
Glucose metabolism. Figure 5 shows time courses for BG and GIR involving treatment with or without wortmannin and insulin. Wortmannin increased BG above basal after ~140 min, but this returned to baseline values at subsequent time points (Fig. 5A). When insulin was infused during clamp conditions, BG was maintained at basal preclamp values by glucose infusion (Fig. 5B). Figure 5C shows time course data for GIR; wortmannin inhibited 10 mU·min–1·kg–1 insulin-mediated glucose requirement, although this took ~1 h to become fully entrenched. Values for GIR at 120 min are shown in Fig. 6A, where it can be seen that both doses of 10 and 20 mU·min–1·kg–1 caused marked increases in glucose requirement with values of 120 ± 5 and 200 ± 4 µmol·min–1·kg–1, respectively. Ra and Rd are shown in Fig. 6, B and C, respectively. Ra was significantly inhibited by both doses of insulin. Wortmannin alone significantly increased Ra ~30%, and, when added with 10 mU·min–1·kg–1 insulin, wortmannin prevented the insulin-mediated reduction of Ra. Rd was increased by both doses of insulin with increases of ~72 and 144 µmol·min–1·kg–1 for 10 and 20 mU·min–1·kg–1 insulin, respectively (Fig. 6C). Wortmannin alone also significantly increased Rd, and wortmannin plus 10 mU·min–1·kg–1 insulin was not significantly different from wortmannin alone. However, the combination of wortmannin plus 10 mU·min–1·kg–1 insulin was lower than 20 mU·min–1·kg–1 insulin alone (Fig. 6C), even though the plasma insulin levels for these two situations were similar (Table 1). HLGU followed a similar pattern to Rd with each dose of insulin stimulatory and that of 20 about two times that of 10 mU·min–1·kg–1 insulin (Fig. 6D). Wortmannin alone was also stimulatory, but the combination of wortmannin plus 10 mU·min–1·kg–1 insulin was lower than 20 mU·min–1·kg–1 insulin alone (Fig. 6D).


Figure 5
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  		Fig. 5. Time course for the effect of wortmannin on blood glucose (BG) in control (saline) and insulin infusions and for whole body glucose infusion rate (GIR) to maintain euglycemia in insulin clamps. Details are as given in Fig. 1. Values are means ± SE for saline ({circ})-, wortmannin (bullet)-, insulin (10 mU·min–1·kg–1, {square})-, insulin (20 mU·min–1·kg–1, {blacksquare})-, and wortmannin + insulin (10 mU·min–1·kg–1, {blacktriangledown})-treated rats.

 

Figure 6
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  		Fig. 6. Effect of wortmannin, insulin, and a combination of wortmannin + insulin on GIR (A), glucose appearance (Ra; B), glucose disposal (Rd; C), and hindleg glucose uptake (HLGU; D). Details are as given in Fig. 1. Values were determined at the end of the experiment (t = 120 min) and are means ± SE for vehicle-treated rats with either saline- or 10 mU·min–1·kg–1 insulin (open bars)-, 20 mU·min–1·kg–1 insulin (gray bars)-, and wortmannin-treated rats with either saline or 10 mU·min–1·g–1 insulin (filled bars). *Insulin-treated significantly different (P < 0.05) from corresponding saline-treated values. #Wortmannin-treated significantly different from corresponding vehicle-treated values. {dagger}20 mU·min–1·kg–1 insulin significantly different from 10 mU·min–1·kg–1 treated.

 
Akt phosphorylation. Figure 7 shows data for Akt phosphorylation (P-Akt/Akt) and Akt total in liver, muscle, and aorta determined by Western blot using antibody to the phospho-Ser473 motif and antibody to Akt. Expression of the results as the ratio P-Akt/Akt shows that insulin in the absence of wortmannin increased the relative proportion of Akt that was phosphorylated in all tissues. Wortmannin inhibited the insulin-mediated increase in P-Akt/Akt in the liver and aorta but not in the skeletal muscle.


Figure 7
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  		Fig. 7. Effect of wortmannin, insulin, and a combination of wortmannin + insulin on phosphorylation of Akt in liver (A), skeletal muscle (B), and aorta (C). Details are as given in Fig. 1 and MATERIALS AND METHODS. Ratio of P-Akt to Akt total has been determined by image analysis of gel bands for vehicle-treated rats with either saline or 20 mU·min–1·kg–1 insulin (open bars) and wortmannin-treated rats with either saline or 10 mU·min–1·kg–1 insulin (filled bars). *Insulin-treated significantly different (P < 0.05) from corresponding saline-treated values. #Wortmannin-treated significantly different from corresponding vehicle-treated values.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study represents the first report of the use of wortmannin to assess effects on insulin action in vivo and particularly to test whether insulin's control of capillary recruitment in muscle is wortmannin sensitive. The study was undertaken because the hemodynamic effects of insulin to increase limb blood flow and capillary recruitment cannot be studied in vitro. The findings show that systemically administered wortmannin inhibited the hemodynamic effects of insulin, suggesting that both limb blood flow and capillary recruitment are controlled by an insulin-signaling cascade involving PI 3-kinase, the target for wortmannin inhibition.

An unexpected effect of wortmannin was the marked increase in plasma insulin without a preceding rise in BG concentration. This rise in plasma insulin produced by wortmannin alone was such that at steady state the plasma levels were ~5,000 pM. Because these levels were similar to those produced by infusion of 20 mU·min–1·kg–1 insulin alone or wortmannin + 10 mU·min–1·kg–1 insulin, comparisons were made with the 20 mU·min–1·kg–1 group rather than the 10 mU·min–1·kg–1 group to assess the effect of wortmannin. From such comparisons, it was clear that the insulin effect to increase either FBF or capillary recruitment was completely inhibited by wortmannin. It was also noteworthy that at the higher plasma insulin concentration generated by infusion of 20 mU·min–1·kg–1 insulin, GIR was further increased above that of 10 mU·min–1·kg–1, but essentially none of this increase was evident at the same high level of plasma insulin (~5,000 pM) when wortmannin was present with 10 mU·min–1·kg–1 insulin. A similar outcome was evident when the data for Rd and HLGU were examined and 20 mU·min–1·kg–1 insulin and wortmannin + 10 mU·min–1·kg–1 insulin were compared.

The finding that wortmannin had a marked effect to increase plasma insulin 10- to 20-fold basal levels most likely relates to an effect on insulin secretion. Explanation for this response is not clear from previous in vitro experiments, since wortmannin has been found by others to both inhibit (4) and stimulate (19, 41, 42) glucose-mediated insulin release by isolated beta-cells. Because a tissue-specific knockout of the beta-cell insulin receptor led to an insulin secretory defect with a loss in insulin release in response to glucose (17), the current findings may suggest that wortmannin in vivo is acting in the pancreas by an insulin receptor-independent pathway.

The effects of wortmannin on metabolic or hemodynamic effects that could be attributed to either liver or the vasculature were generally marked and greater than those associated with muscle. Thus wortmannin fully inhibited insulin-mediated changes in Ra (principally liver), liver P-Akt/Akt, FBF (muscle vasculature), and 1-MX metabolism (muscle vasculature) but only partly inhibited Rd (principally muscle), HLGU, and muscle P-Akt/Akt. Although wortmannin alone markedly increased plasma insulin, aorta P-Akt/Akt was not stimulated, suggesting that wortmannin had completely blocked this effect of insulin. The different responses to wortmannin by liver and aorta compared with the muscle may result from relatively lower delivery to muscle of wortmannin. In liver, the endothelial barrier is incomplete, and delivery of wortmannin, a hydrophobic sterol-like compound, to the liver plasma membrane insulin receptor signaling cascade would be relatively unrestricted with the expectation that insulin-mediated inhibition of Ra would be blocked. Similarly, it is likely that unrestricted delivery of wortmannin to vascular endothelial cells of aorta and muscle vasculature could explain the marked inhibition of insulin-mediated increases in aorta P-Akt/Akt, FBF, and capillary recruitment.

There is substantial evidence that insulin-mediated increase in limb blood flow is nitric oxide dependent (27, 28, 31) and that insulin-mediated nitric oxide production involves Akt-mediated phosphorylation and activation of endothelial nitric oxide synthase (18). Insulin-mediated capillary recruitment is less well defined, but it is plausible that capillary recruitment is controlled at terminal arterioles where insulin receptor tyrosine kinase on endothelial cells initiates a cascade involving insulin receptor substrate-1, PI 3-kinase, Akt, and the phosphorylation and activation of endothelial nitric oxide synthase. There is evidence from cultured endothelial cells to support such a relationship (18). In addition, insulin-mediated capillary recruitment in the human forearm appears to be a locally mediated response (8). Thus the present findings add support to the concept that insulin mediates perfusion of the muscle microvasculature by local control at the terminal arterioles and that this is susceptible to inhibition by wortmannin at the step involving PI 3-kinase.

The finding that indexes of insulin action in muscle such as Rd, HLGU, and muscle P-Akt/Akt were only partly blocked by wortmannin may in part reflect the constraints imposed by the muscle vasculature on delivery of wortmannin to the interstitium and myocyte insulin receptor signaling cascade. Blockade of insulin-mediated capillary recruitment would reduce delivery not only of insulin and glucose but also wortmannin. Reduced delivery of insulin and glucose would give rise to a reduced Rd and HLGU. Based on previous studies where insulin-mediated capillary recruitment has been blocked by a 5-hydroxytryptamine agonist (25), tumor necrosis factor-{alpha} (40), free fatty acids (7), or glucosamine (34), this could amount to ~50% inhibition of the insulin-mediated increase in muscle glucose uptake, with the expectation of a similar magnitude of inhibition of Rd and muscle P-Akt/Akt. In addition to this, it appears likely that the relatively water-insoluble wortmannin is unable to fully diffuse to those myocytes that are unaffected by capillary recruitment (i.e., where insulin delivery is unrestricted). Consequently, a reduced diffusion of wortmannin to these myocytes would limit the extent of inhibition of insulin action, and thus, for the combination of wortmannin + insulin HLGU, Rd and muscle P-Akt/Akt were not fully inhibited compared with liver (Figs. 6 and 7).

Finally, it is important to note that this study is predicated on the assumption that wortmannin at the dose used has specifically inhibited only PI 3-kinase of the insulin-signaling cascade. However, nonspecific effects of wortmannin cannot be entirely ruled out. Low nanomolar concentrations (5–100 nM) are considered to be selective for PI 3-kinase inhibition (11). In vivo, a single bolus injection of 1 mg wortmannin per kilogram body weight was considered by Davol et al. (11) to give a similar outcome in reducing implanted tumor cell volumes as noted for 5–100 nM wortmannin in vitro. The cumulative dose of a constant infusion of 1 µg·min–1·kg–1 wortmannin used in the present study was only 600 µg/kg spread over the 120-min infusion period and thus likely to be somewhat lower than the peak concentration achieved by Davol et al. (11) following a bolus dose of 1 mg. In isolated beta-cells, the effect of wortmannin has been reported to occur at concentrations as low as 10 nM (42) and at 50 nM gave similar results to 10 µM LY-294002, another PI 3-kinase specific inhibitor (42). In muscle, wortmannin inhibits insulin-mediated glucose uptake in either perfused rat hindlimb (36) or isolated incubated epitrochlearis muscles (39) with a half-maximum of ~10 nM. Less specific effects such as the inhibition of contraction-induced glucose uptake are either absent (1 µM; see Ref. 39) or require even higher concentrations of wortmannin (e.g., 3 and 10 µM; see Ref. 36). A recent study by Wang et. al. (35) reported the vascular effects of much larger bolus doses (1,000–7,000 µg/kg) of wortmannin. In that study, wortmannin caused a reduction in BP at the high doses because of inhibition of myosin light-chain phosphorylation. In contrast, in our study with a lower dose of wortmannin, there was a modest increase in BP and femoral VR. Thus, in the present study where the peak wortmannin concentration was likely to have been <100 nM, nonspecific effects were likely to be minimal. The causes for the increase in BP and VR by wortmannin are unknown but may be the result of inhibition of PI 3-kinase-specific vasodilatory action at the peripheral resistance sites or because of central nervous system actions of wortmannin. It is interesting to note that the changes in femoral VR parallel changes in plasma insulin levels caused by wortmannin (Figs. 2 and 3, G and H). Insulin has been shown to cause both nitric oxide and endothelin release from the muscle vasculature, but only the nitric oxide release is PI 3-kinase dependent (14). Thus, in the presence of wortmannin, the increased plasma insulin and associated endothelin release may be the cause for the increase in VR and contribute to the raised BP (21).

In summary, wortmannin infused in vivo at concentrations likely to specifically inhibit PI 3-kinase had profound effects on insulin release and insulin action. Plasma insulin levels increased markedly, and the hemodynamic effects of insulin of increased limb blood flow and capillary recruitment were completely inhibited, as was the insulin-mediated inhibition of hepatic glucose output. Delivery of wortmannin to the myocytes may be restricted, and insulin-mediated glucose uptake and Rd were only partly inhibited, but this may be the result of reduced delivery of insulin and glucose because of the total inhibition of insulin-mediated capillary recruitment.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by the National Health and Medical Research Council, Australian Research Council, and the Heart Foundation of Australia. S. Rattigan is a Heart Foundation Career Fellow.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Rattigan, Biochemistry, Private Bag 58, Univ. of Tasmania, Hobart, 7001 TAS, Australia (e-mail: S.Rattigan{at}utas.edu.au)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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