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Basic Science Aspects

BLOCKADE OF THE RENIN-ANGIOTENSIN SYSTEM IMPROVES INSULIN SENSITIVITY IN THERMAL INJURY

Kasper, Sherry O.; Castle, Scott M.; Daley, Brian J.; Enderson, Blaine L.; Karlstad, Michael D.

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doi: 10.1097/01.shk.0000230302.24258.9f
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Abstract

INTRODUCTION

Insulin resistance is recognized as a major contributing factor to morbidity and mortality in burn injury, with a large percentage of patients developing hyperglycemia and glucose intolerance (1-3). Tight plasma glucose control in both sepsis (4) and burn injury (5-9) has proven to be important for the healing and survival of the injured animal or patient. The mechanisms underlying hyperglycemia have not been fully characterized, but there are several possibilities including an increase in gluconeogenesis and a decrease in insulin-dependent glucose uptake. In fact, rats with a severe burn injury have a decrease in skeletal muscle glucose transport in response to insulin treatment as compared with sham-burned animals. In addition, these animals have a decrease in insulin signaling including a decrease in insulin receptor substrate 1 (IRS-1) activation and a decrease in phosphatidylinositol 3 kinase (PI 3-K) activity (9).

The renin-angiotensin system (RAS) has been recognized as an important contributor to insulin resistance and type II diabetes, especially in patients with essential hypertension (10). In several clinical trials, angiotensin-converting enzyme (ACE) inhibitors and angiotensin II type 1 (AT1) receptor blockers reduced the risk of developing type II diabetes (11, 12). Not only does treatment with a RAS blocker reduce the risk of developing type II diabetes, but it also is important in improving glucose use. Patients treated for 4 months with an ACE inhibitor, captopril, showed improvements in the disposal of glucose as compared with placebo, whereas a diuretic (hydrochlorothiazide) decreased glucose disposal (13). Also, in obese Zucker rats, a model of type II diabetes, administration of an AT1 receptor blocker improved insulin sensitivity and glucose transport activity and glucose transporter 4 (Glut4) protein level in muscle (14). In another rat model of insulin resistance, streptozotocin-induced type 1 diabetes, valsartan, an AT1 receptor blocker, lowered plasma glucose and improved insulin resistance measured by the intravenous glucose challenge test. In addition, valsartan also increased the messenger RNA and protein levels of Glut4 (15). The AT1 receptor blockade also lowers insulin resistance in sucrose-fed spontaneously hypertensive rats and Wistar Kyoto (16). It has been shown that blockade of the AT1 receptors may improve insulin signaling by interrupting the inhibitory effects of angiotensin II on PI 3-K and Glut4 in insulin-resistant obese Zucker rats (14). The PI 3-K is considered to be essential for insulin-stimulated Glut4 translocation in skeletal muscle. These studies suggest a significant role of RAS in the modulation of insulin resistance.

The RAS also seems to play a critical role in the pathophysiology of burn injury. The production and plasma levels of angiotensin II are increased in burn injury (17, 18). Because RAS is upregulated in burn injury, it is important to investigate the role of RAS in the development of insulin resistance in burn injury. Thus, interruption of RAS may be a mechanism for improving insulin sensitivity in burn injury. Our goal is to elucidate the regulatory role of RAS in the development of insulin resistance in burn injury, which may lead to new insights into glucose homeostasis and/or new therapies for burn injury.

METHODS

Animals

Male Sprague Dawley rats were ordered from Taconic Farms (Germantown, NY) at 200-225 g. They were allowed to habituate to our animal facilities for 7 days before burn or sham-burn treatment. Animals were exposed to the same housing conditions (light/dark cycle of 12:12) and provided with food and water ad libitum. These rats were used at 250-270 g. Animals were killed at the end of the study, and trunk blood was taken for further analysis. The Animal Care and Use Committee of the University of Tennessee approved the experimental protocols in accordance with the Institute for Laboratory Animal Research Guide for Care and Use of Laboratory Animals.

Thermal injury

Animals were fasted for 16 h and then anesthetized with 3% isoflurane and exposed to a 30% body surface area full thickness third degree burn by immersion of the dorsum of the animal through a preformed mold into water with a temperature level of 95°C for 15 s (19, 20). Sham animals were anesthetized and exposed to lukewarm water (23°C) in the same manner as the burned animals. Lactated Ringer's solution (2.5 mL/kg) was given subcutaneously immediately after the burn and sham injury for fluid resuscitation. The AT1 receptor blocker, losartan, (Merck & Co, Inc, Rahway, NJ) was diluted in water (1 mL) and administered at a dosage of 30 mg/kg per day by gavage immediately after the burn injury and daily for 3 subsequent days (21-24). This dosage of losartan was used because of previous studies suggesting improved cardiovascular and glucose metabolism in treated animals and no long-term adverse effects. Sham-burned and burn placebo animals received water (1 mL) by gavage immediately after the sham or burn injury and daily for 3 days. There is clear insulin resistance in this thermal injury model 3 days after burn injury (9).

Oral glucose tolerance test

Animals were given 1 g/kg dextrose by gavage, and blood was taken before and 15, 30, 60, and 90 min after the gavage from a clip at the end of the tail (14). This procedure was done between 11:00 am and 1:00 pm after a 16-h fast. Area under the curve (AUC) was determined for both insulin and glucose. Glucose-insulin index was calculated as AUC insulin × AUC glucose (14).

Radioimmunoassays and glucose determination

Insulin levels were measured by radioimmunoassay (Linco Research, Inc, St. Charles, Mo). Glucose was measured using a FreeStyle glucose analyzer (TheraSense, Inc; Alameda, CA), as done previously (25). Plasma angiotensin II levels were measured by radioimmunoassay (ALPCO Diagnostics, Salem, NH/USA), as previously described (21).

Statistics

One-way analysis of variance was performed between groups with Tukey post hoc test. A P < 0.05 was used to determine significance.

RESULTS

The insulin and glucose curves from the oral glucose tolerance test cure shown in Figure 1. As demonstrated in Figure 2A, there was an increase in the AUC for insulin because of burn injury, and this increase was abolished with losartan treatment. There was no difference in AUC for glucose among the groups (Fig. 2B). Burned animals have a higher glucose-insulin index, a measure of whole body insulin sensitivity, compared with both the sham-burned control and burned animals treated with losartan (P < 0.05). Losartan treatment returns the glucose-insulin index to the level of the sham-burned animals (Fig. 3). Also, as demonstrated in Table 1, there was no difference in body weight between burned and sham-burned control animals or between those burned animals receiving losartan or placebo. Finally, there was no difference in plasma angiotensin II levels in sham or burned animals 3 days postburn injury. However, losartan treatment resulted in a significant (P < 0.05) increase in plasma angiotensin II levels (Table 1), as expected from previous reports (21).

Fig. 1
Fig. 1:
Insulin and glucose levels before and at 15, 30, 60, and 90 min postglucose administration during the oral glucose tolerance test in sham, burn placebo, and burn losartan rats 3 days postburn injury; n = 5 to 6 in each group.
Fig. 2
Fig. 2:
Area under the curve for insulin and glucose taken during the oral glucose tolerance test in sham, burn placebo, and burn losartan rats 3days postburn injury. A, AUC for insulin. Burn placebo animals have an increase in AUC insulin compared with sham-burned animals, and losartan treatment abolished this increase. B, AUC for glucose. There is no difference among groups; n = 5 to 6 in each group. *P < 0.05 versus other groups. BL indicates burn losartan; BP, burn placebo.
Fig. 3
Fig. 3:
Glucose-insulin index in sham, burn placebo, and burn losartan animals 3 days postburn injury. Burn placebo animals have an increase in the glucose insulin index compared with sham-burned animals; losartan treatment returned this index to normal. *P < 0.05 versus other groups; n = 5 to 6 in each group. Abbreviations are explained in the legend to Figure 2.
Table 1
Table 1:
Body weight and plasma angiotensin II levels for sham, burn placebo, and burn losartan animals

DISCUSSION

This study demonstrates that RAS plays a major role in the insulin resistance induced by burn injury. The AT1 receptor blockade by losartan treatment abolished this insulin resistance as measured by the oral glucose tolerance test. We show that burned animals have a higher AUC for insulin and a higher glucose insulin index compared with the sham-burned control animals. The AT1 receptor blockade abolished the rise in these parameters of insulin resistance, indicating complete reversal of the insulin resistance of burn injury. In fact, there was no difference in AUC for insulin or the glucose-insulin index between the sham-burned animals and the burned animals treated with losartan. Although plasma angiotensin II levels are not elevated 3 days after burn injury, there is evidence that activation of this system occurs 12 to 24 h after burn injury (18). Also, because there are independent RAS systems in the skeletal muscle (26), adipose tissue (27), and pancreas (28), RAS may be elevated in these systems, resulting in insulin resistance during burn injury. Therefore, RAS may prove to be a new therapeutic target for the treatment of insulin resistance associated with burn injury.

Burn injury is associated with hyperglycemia and insulin resistance in patients with no previous signs of this pathology (9). It was once accepted that this rise in blood glucose was necessary for the hypermetabolic state that accompanies critical illness. However, in a recent study, intensive insulin therapy, which consisted of maintaining blood glucose at or below 110 mg/dL, was compared with conventional therapy, which consisted of maintenance of blood glucose at or below 215 mg/dL, in critically ill patients. Remarkably, intensive insulin therapy resulted in 32% reduction in mortality rate, 46% reduction in septicemia, and reduced the length of stay in the intensive care unit (29). Also, in a rabbit model of critical illness, control of hyperglycemia prevented excessive inflammation, weight loss, and the impairment of insulin release caused by burn injury (5). Finally, in severely burned pediatric patients, intensive insulin therapy improved the survival and infection rate (30). Therefore, control of hyperglycemia is important to the morbidity and mortality of burn-injured and critically ill patients.

In the present study, insulin resistance was evident in the burned animals compared with the sham-burned control animals, as expected from previous reports (9, 31). Many changes in postreceptor insulin signaling occur during burn injury that result in insulin resistance including a decrease in PI 3-K activity, IRS-1 tyrosine phosphorylation, (9) and Akt/protein kinase B serine phosphorylation (31). In fact, in a rat model of burn injury, glucose uptake into muscle strips was reduced by 30% compared with sham-burned animals, and insulin-stimulated glucose uptake was completely abolished (9). Insulin-stimulated PI 3-K activity was also diminished in the burn-injured animals including the tyrosine phosphorylation of IRS-1 and the IRS-1/PI 3-K association (9). In a model of burn injury in mice with 25% total body surface area burn, there was an increase in IRS-1 phosphorylation at the serine 307, which is inhibitory to insulin signaling, in the burn animals. In this model, total IRS-1 including Akt/protein kinase B activity was decreased with burn injury (31). Therefore, many postreceptor modifications occur in burn injury that affect insulin resistance and may be important to the insulin resistance observed in our burn model.

Treatment with an ACE inhibitor or AT1 receptor blocker improves insulin sensitivity both in animal models (14, 32, 33) and in humans (10, 12). Large clinical trials have shown that treatment with RAS inhibitors decreases the risk of the development of type II diabetes including Heart Outcomes Prevention Evaluation, Captopril Prevention Project, Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial, and several others (12). In addition, blockade of the RAS improves glucose use in many animal models of insulin resistance such as type II diabetic KK-Ay mice (32), obese Zucker rats (14), sucrose fed rats (16), and streptozotocin-induced diabetic rats (15). Also, impaired glucose use and insulin signaling occur in animals with high expression of RAS [(mRen2)27] (25, 34); animals with low brain RAS have improved glucose use compared with Sprague Dawley rats (25). However, the contribution of RAS to the burn injury-induced insulin resistance is unknown. We show (Fig. 3) for the first time that losartan treatment completely abolished the insulin resistance that occurs with burn injury.

There are many possible mechanisms for the beneficial effects of RAS blockade on insulin sensitivity. Angiotensin II infusion both in vivo and in vitro inhibits insulin signaling by attenuating PI 3-K activity in response to insulin in the intact rat heart and by inducing serine phosphorylation of IRS-1 and IRS-2 in rat aorta smooth muscle cells (35). The (mRen2)27 rats with increased expression of RAS exhibit a reduction in tyrosine phosphorylation of the insulin receptor and IRS-1 in addition to an inhibition of the association between PI 3-K and IRS-1. Also, serine phosphorylation of Akt was diminished in the (mRen2)27 rats compared with Sprague Dawley rats. Conversely, treatment with an AT1 receptor blocker improves tyrosine phosphorylation of IRS-1 and insulin receptor IRS-1/PI 3-K association, and Glut4 translocation to the plasma membrane. Valsartan treatment improves PI 3-K activity in diabetic KK-Ay mice (32), and irbesartan treatment increases total Glut4 in obese Zucker rats (14). Finally, angiotensin II treatment inhibits Akt activation in vascular smooth muscle cells (36). These data suggest that there is a significant negative cross talk between the AT1 receptor and the insulin receptor. Many of these same alterations occur in burn injury as well, suggesting that angiotensin II may be responsible for these alterations in insulin signaling.

Because RAS activation during burn injury occurs to maintain blood pressure and homeostasis, blockade of this system may decrease the chance for survival. However, there were no deaths in our study. In fact, several studies suggest that blockade of RAS is beneficial to burn-injured animals. Treatment with DuP753, an AT1 receptor blocker, decreases bacterial translocation, mucosal permeability, and gut ischemia caused by burn or endotoxin injury (37) and improves hepatic blood flow after burn injury (38). Treatment with an ACE inhibitor, enalapril, actually improved survival rates in burn injured mice and decreased bacterial translocation (39). Therefore, administration of losartan to burn-injured animals does not seem to cause detrimental effects.

These data indicate that RAS is crucial in the development of insulin resistance associated with burn injury. Blockade of this system resulted in complete reversal of insulin resistance in our studies. Therefore, RAS may be an important therapeutic target for the treatment of burn injury in the future. However, further studies need to be performed to determine the precise mechanism.

ACKNOWLEDGMENTS

The authors thank Merck & Co, Inc, Rahway, New Jersey for the generous gift of losartan potassium.

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Keywords:

Insulin resistance; burn; rat; losartan; AT1 receptor; renin-angiotensin system

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