α-Adrenergic Stimulation Mediates Glucose Uptake Through Phosphatidylinositol 3-Kinase in Rat Heart
Abstract—We examined whether insulin and catecholamines share common pathways for their stimulating effects on glucose uptake. We perfused isolated working rat hearts with Krebs-Henseleit buffer containing [2-3H]glucose (5 mmol/L, 0.05 μCi/mL) and sodium oleate (0.4 mmol/L). In the absence or presence of the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin (3 μmol/L), we added insulin (1 mU/mL), epinephrine (1 μmol/L), phenylephrine (100 μmol/L) plus propranolol (10 μmol/L, selective α-adrenergic stimulation), or isoproterenol (1 μmol/L) plus phentolamine (10 μmol/L, selective β-adrenergic stimulation) to the perfusate. Cardiac power was found to be stable in all groups (between 8.07±0.68 and 10.7±0.88 mW) and increased (25% to 47%) with addition of epinephrine, but not with selective α- and β-adrenergic stimulation. Insulin and epinephrine, as well as selective α- and β-receptor stimulation, increased glucose uptake (the following values are in μmol/[min · g dry weight]: basal, 1.19±0.13; insulin, 3.89±0.36; epinephrine, 3.46±0.27; α-stimulation, 4.08±0.40; and β-stimulation, 3.72±0.34). Wortmannin completely inhibited insulin-stimulated and selective α-stimulated glucose uptake, but it did not affect the epinephrine-stimulated or selective β-stimulated glucose uptake. Sequential addition of insulin and epinephrine or insulin and α-selective stimulation showed additive effects on glucose uptake in both cases. Wortmannin further blocked the effects of insulin on glycogen synthesis. We conclude that α-adrenergic stimulation mediates glucose uptake in rat heart through a PI3-K–dependent pathway. However, the additive effects of α-adrenergic stimulation and insulin suggest 2 different isoforms of PI3-K, compartmentation of PI3-K, potentiation, or inhibition by wortmannin of another intermediate of the α-adrenergic signaling cascade. The stimulating effects of both the α- and the β-adrenergic pathways on glucose uptake are independent of changes in cardiac performance.
Glucose uptake in heart muscle is stimulated by insulin and catecholamines.1 2 3 It is not known whether the signaling pathways for the hormones are separate or share common intermediates. Stimulation with insulin results in receptor autophosphorylation and subsequent activation of a plethora of mediators, among them insulin receptor substrate 1, which activates phosphatidylinositol 3-kinase (PI3-K). PI3-K has been consistently shown to mediate the membrane-trafficking effects of insulin leading to increased glucose uptake by recruitment of glucose transporters from an intracellular pool.4 5 6 7 The PI3-K family of enzymes is known to be regulated by several different upstream pathways in response to virtually all growth factors and cytokines and can be inhibited by the fungal metabolite wortmannin.8
Several hypotheses have been advanced for the stimulating effects of catecholamines on glucose uptake. These hypotheses include the stimulation of glucose transporter recruitment from the same or a different pool of intracellular transporters by contraction, increases in intracellular [Ca2+], or the activation of the α-adrenergic pathway.9 10 11 12 Recent evidence suggests phosphorylation of insulin receptors by components of the α-adrenergic pathway,13 and the involvement of α-adrenergic activation in the mechanism of pressure overload hypertrophy14 suggests the involvement of the α-adrenergic pathway in growth signaling. Thus, it is conceivable that the signaling cascades activated by the different interventions, leading to increased glucose uptake in heart, may overlap considerably.
We examined whether insulin and catecholamines stimulate glucose uptake in heart muscle by the same or by different mechanisms. We found that α-adrenergic stimulation mediates glucose uptake through a PI3-K–dependent pathway. Because of the additive effects of α-adrenergic stimulation and insulin, we conclude that there are 2 different isoforms of PI3-K, there is compartmentation of PI3-K, there is potentiation of the insulin-receptor and the α-receptor pathways, or there is inhibition by wortmannin of another intermediate of the α-adrenergic signaling cascade, possibly protein kinase C.
Materials and Methods
Male Sprague-Dawley rats (300 to 350 g) were obtained from Harlan (Indianapolis, IN). Animals were fasted overnight (16 to 20 hours) with free access to water. The use of animals and the experimental protocol were approved by the Animal Welfare Committee of the University of Texas-Houston Health Science Center.
Chemicals were obtained from Fisher Scientific or Sigma. Enzymes and cofactors for metabolite assays were obtained from Boehringer Mannheim or Sigma Chemical Co. Regular human insulin (Humulin R) was purchased from Eli Lilly and Co (Indianapolis, IN). Wortmannin and the catecholamines were from Sigma.
HPLC-purified [2-3H]glucose was obtained from Amersham. Before each perfusion we ascertained that 3H radioactivity was in the tracer and not in 3H2O. The tracer was discarded if the activity from intrinsic tritiated water was >1% of the total activity. A purity check of the tracer was also performed using the method described by Cheung et al15 to ascertain that the tracer was indeed [2-3H]glucose.
Working Heart Preparation
The preparation has been described in detail earlier.16 Briefly, rats were anesthetized with sodium pentobarbital (5 mg/100 g body weight IP). After injection of heparin (200 IU) into the inferior vena cava, the heart was rapidly removed and placed in ice-cold Krebs-Henseleit buffer. The aorta was cannulated, and a brief period of retrograde perfusion (<5 minutes) with oxygenated buffer containing glucose (5 mmol/L) was performed to wash out any blood from the heart and to cannulate the left atrium. Hearts were then perfused as working hearts at 37°C with recirculating Krebs-Henseleit buffer (200 mL) containing glucose (5 mmol/L) and sodium oleate (0.4 mmol/L) bound to 1% BSA and Cohn fraction V (fatty acid free; Intergen Co) and equilibrated with 95% O2-5% CO2. Perfusate [Ca2+] was 2.5 mmol/L. All experiments were carried out with a preload of 15 cm H2O and an afterload of 100 cm H2O. The hearts were beating spontaneously at an average rate of 300 bpm. Aortic flow and coronary flow were measured every 5 minutes, and cardiac output was calculated as the sum of both values. Heart rate and systolic and diastolic aortic pressures were measured continuously with a 3F Millar transducer (Millar Instruments) and a MacLab physiological recording system (ADInstruments).
Hearts were perfused for 60 or 90 minutes in the presence or absence of wortmannin (3 μmol/L). At a concentration of 3 μmol/L, wortmannin completely inhibited insulin-stimulated glucose uptake. Wortmannin was dissolved as a 10 mmol/L solution in DMSO freshly on the day of every experiment. [2-3H]Glucose (10 μCi) was added to the perfusate (200 mL) before the beginning of the experiment. Table 1⇓ shows the different experimental groups and the interventions, which were performed at t=30 minutes (and t=60 minutes in 2 groups). The concentrations of insulin (1 mU/mL), epinephrine (1 μmol/L), isoproterenol (1 μmolL), and phenylephrine (100 μmol/L) were chosen to achieve maximal stimulatory effects. The concentrations of propranolol (10 μmol/L) and phentolamine (10 μmol/L) were chosen because they fully inhibited the effects of isoproterenol and phenylephrine, respectively (data not shown). Hearts in the Alpha and Alpha-WM groups and Beta and Beta-WM groups were stimulated with an α-adrenergic agonist plus a β-adrenergic antagonist and a β-adrenergic agonist plus an α-adrenergic antagonist, respectively (hearts in the Alpha-WM and Beta-WM groups were treated with wortmannin). This approach was chosen for the following 2 reasons. First, the presence of an inhibitor of the receptor type not to be stimulated eliminates any cross-stimulation by endogenous catecholamines. Secondly, the negative inotropic effects of α- and β-blockers blunted the positive inotropic effects of the α- and β-adrenergic agonists (data not shown). At the end of all perfusions, hearts were freeze-clamped with aluminum tongs cooled to the temperature of liquid nitrogen.
Assessment of Contractile Performance
Mean aortic pressure (cm H2O) was calculated as (systolic+diastolic pressure×2)/3. Heart rate was measured as bpm and cardiac output as mL/min. Cardiac power was determined from the product of cardiac output and mean aortic pressure as described earlier.17
Measurement of Glucose Uptake
Glucose uptake was determined by the rate of 3H2O production from [2-3H]glucose.18 Counting of the isotope was performed on a Packard 1900 TR liquid scintillation analyzer by the method of spectral index analysis as described by the manufacturer (Packard Instruments). Release of 3H2O into the perfusate was analyzed in 5-minute intervals. 3H2O was separated from [2-3H]glucose in the perfusate by anion exchange chromatography on AG-1X8 resin (Bio-Rad Laboratories).19 The amount of 3H2O in the perfusate was plotted against time, and the slopes of the desired intervals were used to calculate glucose uptake rates, which were expressed as μmol/(min · g dry weight).
The frozen tissue, ground under liquid nitrogen, was extracted with 6% perchloric acid. The tissue extracts were neutralized with 3 mol/L KOH. A small portion of the pulverized tissue was dried in an oven (70°C) to constant weight and the wet-to-dry ratio was calculated. The tissue extracts were analyzed for glycogen as reported by Walaas and Walaas20 and for glucose-6-phosphate (G6P) and lactate content according to standard biochemical methods.
Samples of the coronary effluent (1 mL) were withdrawn every 5 minutes and were stored on ice until assayed for glucose and lactate by a glucose/lactate analyzer (2300 STAT, YSI Inc). The samples were analyzed for the specific activity ([2-3H]glucose) and for 3H2O content.
All data are presented as mean±SEM. Statistical comparison was by 1-way repeated-measures analysis or unpaired ANOVA with post hoc comparison by Newman-Keuls test as appropriate.21 Differences were considered statistically significant when P<0.05.
Cardiac power was stable in all groups before and after the interventions (Table 2⇓). Cardiac power in the first 30 minutes ranged from 8 to 11 mW. Except for epinephrine, which caused a 25% to 47% sustained increase in cardiac power, none of the interventions changed contractile performance, and cardiac power remained stable for the entire duration of the experiments. Although the response to epinephrine was less pronounced in the presence of wortmannin, the differences were not statistically significant. In preliminary experiments, it was established that the positive inotropic responses of the α- and the β-adrenergic agonists were blocked by the α- and β-adrenergic antagonists, respectively (data not shown).
Figure 1⇓ shows rates of myocardial glucose uptake before and after the addition of insulin without or with wortmannin. Insulin addition caused a 3-fold increase in glucose uptake that was completely inhibited by wortmannin (shaded bars).
Figure 2⇓ shows rates of myocardial glucose uptake before and after the addition of epinephrine in the presence or absence of wortmannin. Epinephrine, as insulin, caused a significant increase in glucose uptake, but this increase was not inhibited by wortmannin.
To distinguish between the α- and β-receptor–mediated effects of epinephrine, we stimulated hearts in the presence or absence of wortmannin with either phenylephrine or isoproterenol. To eliminate interference from endogenous catecholamines from cardiac nerve termini, we inhibited the β-receptor with propranolol in hearts in which phenylephrine was added and, conversely, we inhibited the α-receptor with phentolamine in hearts in which isoproterenol was added. Figures 3A⇓ and 3B⇓ demonstrate that the selective stimulation of either α- or β-adrenergic receptors increased glucose uptake to the same degree as epinephrine. However, whereas wortmannin did not affect β-stimulated glucose uptake, the α-stimulated uptake was completely inhibited. The changes in glucose uptake observed with selective α- and β-adrenergic stimulation were similar to the effects of epinephrine, although contractile function was unchanged in these protocols.
Figure 4⇓ shows glucose uptake of those hearts in which the stimulation with insulin was followed by either epinephrine (Figure 4A⇓) or α-selective stimulation (Figure 4B⇓). In both groups the effects of the second intervention were additive to the effects of insulin on glucose uptake, but epinephrine caused a greater increase than selective α-receptor stimulation or epinephrine in the absence of insulin (see Figure 2⇑).
It is of note that wortmannin did not inhibit unstimulated glucose uptake in any of the groups (ie, glucose uptake measured in the absence of insulin or catecholamines [Figures 1 through 3⇑⇑⇑, before intervention]).
Glycogen and Metabolites
Figure 5⇓ shows the change in tissue content of glycogen caused by the different interventions. The total glycogen content before the interventions was 71.0±3.04 μmol/g dry weight. The total glycogen content at the end of the experiments is shown at the bottom of Figure 5⇓ for each group. Addition of insulin increased myocardial glycogen content significantly. This increase was completely inhibited by wortmannin. Addition of epinephrine resulted in significant net glycogen breakdown, which was unaffected by wortmannin. Selective β-receptor stimulation caused glycogen breakdown similar to epinephrine, whereas α-receptor stimulation only caused an insignificant amount of glycogen breakdown. Wortmannin did not affect these changes significantly. Insulin blunted the net breakdown of glycogen induced by epinephrine or α-adrenergic stimulation.
We also measured G6P, which is a regulator of glycogen synthesis. The tissue content of G6P was highest after the addition of insulin (0.91±0.24 μmol/[min · g dry weight]) and lowest after the addition of epinephrine (0.23±0.24 μmol/[min · g dry weight]). Statistical comparison of all 10 groups did not reveal any significant differences.
This study provides evidence that α-adrenergic stimulation mediates glucose uptake in rat heart through a PI3-K–dependent pathway. The observed additive effects of α-adrenergic stimulation and insulin suggest the following possibilities: there are 2 different isoforms of PI3-K, there is compartmentation of PI3-K, there is potentiation of the insulin and the α-receptor pathways, or there is inhibition by wortmannin of another intermediate of the α-adrenergic signaling cascade. The study further demonstrates that the stimulating effects of both the α- and the β-adrenergic pathways on glucose uptake are independent of changes in cardiac performance. Finally, it could be demonstrated that the effects of insulin on glycogen synthesis are also mediated through PI3-K and that the effects of epinephrine on glycogen breakdown are mediated through the β-adrenergic pathway.
Our finding of increased glucose uptake on epinephrine addition is in agreement with other investigations performed in the isolated heart3 9 22 but is in contrast to results obtained by addition of epinephrine in vivo.23 24 The observed decrease in glucose uptake on catecholamine addition in vivo may be due to catecholamine effects on other metabolically active tissues. It is conceivable that the increase in lactate production and release of fatty acids by skeletal muscle on epinephrine addition increases the availability of these substrates to the heart. The preference of the heart for the oxidation of lactate and fatty acids under normoxic conditions22 may then overcome the stimulatory effects of epinephrine on glucose metabolism and result in a net decrease of glucose uptake. These discrepancies of the epinephrine effects in vivo and in vitro demonstrate the importance of isolated systems for the investigation of mechanisms by which hormones elicit their effects on myocardial metabolism. We used the isolated working rat heart preparation for our studies, because the model allows us to eliminate the interference from peripheral tissues in vivo and provides control of the metabolic and hormonal environment, while contractile function is similar to contractile function in vivo. Despite the differences in the effects of catecholamines in vivo and in vitro, it is reasonable to assume that the signal-transduction cascades elucidated in this study are also activated by epinephrine in vivo, in which the effects may be blunted by other regulatory mechanisms.
Inhibition of PI3-K by Wortmannin
The fungal metabolite wortmannin is a potent inhibitor of PI3-K. Wortmannin is thought to be specific at low concentrations.25 26 At a concentration of 3 μmol/L, we found complete inhibition of insulin-induced glucose uptake. This concentration is 30 times the Ki for PI3-K in soleus muscle25 (ie, inhibition of PI3-K can be expected to be near 100%) and is consistent with those used in skeletal muscle, adipocytes, and heart muscle (0.1 to 5 μmol/L).6 7 27 28
Wortmannin has also been shown to inhibit other cellular enzymes such as protein kinase C and myosin light chain kinase, but the concentrations required for these effects were 100- to 1000-fold higher than those necessary to inhibit glucose uptake.25 26 It appears therefore reasonable to assume that the concentration of 3 μmol/L, which we chose for the present study, renders wortmannin a specific inhibitor of PI3-K. This conclusion is consistent with the one drawn from studies in brown adipocytes.29 However, significant inhibition of protein kinase C by wortmannin may be an alternative explanation for the inhibition of α-adrenergically stimulated glucose uptake by wortmannin.
Mechanism of Myocardial Glucose Uptake
Because of their importance for the understanding of type 2 diabetes mellitus, the mechanisms underlying the regulation of glucose uptake by insulin have been intensely investigated and are largely understood.4 30 Activation of the insulin receptor leads to autophosphorylation of the receptor followed by the activation of insulin-receptor substrate 1, which forms a complex with and activates PI3-K, the key mediator for the acute effects of insulin on metabolism.4 5 25 Our findings of the inhibition of insulin-induced glucose uptake and the inhibition of insulin-induced glycogen synthesis are in support of this concept.
In contrast, only little is known about the mechanism responsible for the effects of catecholamines on glucose uptake. It has been suggested that the effects of catecholamines on glucose uptake in skeletal31 32 and in heart muscle9 are contraction mediated (ie, mediated through an increase in cytosolic [Ca2+]). Another hypothesis on the effects of catecholamines on glucose uptake has arisen from observations in cardiac myocytes. In contrast to the contraction-mediated induction of glucose uptake, it has been suggested that α-receptor–coupled G proteins are responsible for the effect, whereas the stimulation of β-receptors does not increase glucose uptake.11 We demonstrate in the isolated working rat heart that specific stimulation of β-receptors increases glucose uptake to the same degree as stimulation of α-receptors. In light of the lack of β-receptor effects in noncontracting cardiac myocytes, it is conceivable that α- and β-receptors stimulate different signaling pathways to increase glucose uptake, in which contraction is required for the effects of the β-receptor pathway. Our results support the concept of different pathways for the stimulation of glucose uptake through α- or β-receptors. The lack of an increase in contractile performance in our protocol of selective α- and β-stimulation demonstrates that an increase in contractile function is not necessary to elicit the stimulatory effects on glucose uptake. However, we did not measure the intracellular concentration of Ca2+.
We demonstrate that a PI3-K inhibitor is able to block a pathway activated by α-adrenergic receptor stimulation. However, despite the complete inhibition of the α-adrenergically stimulated glucose uptake, the effects of selective α-adrenergic stimulation were still additive to the effects of insulin on glucose uptake. There are 4 possible explanations for these observations. First, α-receptor stimulation leads to the activation of a different isoform or a subfraction of PI3-K, which is also inhibited by wortmannin but not involved in the insulin signaling pathway. The existence of different subfractions of PI3-K has already been suggested in skeletal muscle.27 Second, the signaling pathways may be compartmentalized. Third, it is conceivable that the effects of insulin and α-receptor stimulation potentiate each other. It could be speculated that the common link is protein kinase C, which has been shown to increase glucose uptake when stimulated with phorbol esters33 and which is able to phosphorylate the insulin receptor.13 Fourth, wortmannin may not be selective for PI3-K and inhibit a component of the α-adrenergic signaling pathway. However, the specificity of wortmannin has been documented in many studies. Despite the possibility of nonspecific interference of wortmannin with the α-adrenergic pathway, the data strongly support the concept of 2 distinct mechanisms for α- and β-adrenergically stimulated glucose uptake.
The presence of different pools of glucose transporters in the cytosol has been suggested that are thought to be recruited by different mechanisms.34 35 The additive effects of the insulin and epinephrine and insulin and selective α-adrenergic stimulation support this hypothesis. This hypothesis is further supported by our findings that the additive effects of epinephrine and insulin are greater than the additive effects of selective α-stimulation and insulin. Because selective α- or β-receptor stimulation alone reached the same magnitude of glucose uptake as maximal stimulation with epinephrine, one may speculate that the α-receptor and the insulin pathways potentiate each other. This hypothesis would explain the greater response of glucose uptake to epinephrine than to selective α-stimulation in the presence of insulin or to epinephrine alone, because the α-adrenergic component of epinephrine would be amplified by the presence of insulin.
The possibility exists that the additive effects of insulin and epinephrine could be due to an underestimation of insulin-stimulated glucose transport. We have shown before that insulin causes a shift of the rate-limiting step for glucose uptake from transport to phosphorylation36 (ie, insulin stimulates glucose transport more than phosphorylation), and, thus, glucose uptake underestimates glucose transport at this point. If this were the case in the present experiments, then the error would be a systematic error, which would not affect the interpretation of our results. The work of Cheung et al15 showed that glucose uptake as measured by glucose disappearance may be up to 13% higher than uptake measured by 3H2O production from [2-3H]glucose under conditions of high glucose and high insulin concentrations and in the absence of fatty acids. The discrepancy may be due to the same shift of the rate-limiting step of glucose uptake as described above. Even with some underestimation of insulin-stimulated glucose transport by the [2-3H]glucose method, the overall interpretation of our results would be the same.
Myocardial Glycogen Synthesis and Degradation
The involvement of PI3-K in the effects of insulin on glycogen synthesis has been documented in skeletal muscle and adipocytes.25 37 Recently, it has been doubted whether this pathway is dependent on PI3-K.38 If wortmannin is a specific inhibitor of PI3-K, then stimulation of glycogen synthesis in rat heart must be PI3-K dependent.
The key enzyme for glycogen breakdown is glycogen phosphorylase, which is activated by phosphorylase b kinase, a reaction activated by [Ca2+]. cAMP activates phosphorylase b kinase through activation of cAMP-dependent protein kinase. The stronger effect of β-receptor activation compared with the effect of α-receptor activation on glycogen breakdown found in this study suggests that the described mechanism is much more sensitive to cAMP than to [Ca2+] in heart muscle.
We have shown that α-adrenergic stimulation mediates glucose uptake in rat heart through a PI3-K–dependent pathway. The observed additive effects of α-adrenergic stimulation and insulin suggest 2 different isoforms of PI3-K, compartmentation of PI3-K, or inhibition by wortmannin of another intermediate of the α-adrenergic signaling cascade. The study further demonstrates that the stimulating effects of both the α- and the β-adrenergic pathways on glucose uptake are independent of changes in cardiac performance. Finally, we demonstrated that the effects of insulin on glycogen synthesis are also mediated through PI3-K and that the effects of epinephrine on glycogen breakdown are mediated through the β-adrenergic pathway.
This study was supported in part by a grant from the United States Public Health Service (RO1-HL43133). T.D. was the recipient of a research fellowship from the German Research Foundation (Deutsche Forschungsgemeinschaft). We thank Patrick Guthrie for technical assistance, Prof Dr F. Beyersdorf for encouragement, and Drs Gary W. Goodwin and Christophe Depre for helpful discussions.
Presented in part at the 70th Annual Scientific Sessions of the American Heart Association, Orlando, Fla, November 9–12, 1997, and published in abstract form (Circulation. 1997;96[suppl I]:I-691).
- Received July 27, 1998.
- Accepted December 11, 1998.
- © 1999 American Heart Association, Inc.
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