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. 2008 May 1;149(8):4043–4050. doi: 10.1210/en.2007-1646

Captopril Normalizes Insulin Signaling and Insulin-Regulated Substrate Metabolism in Obese (ob/ob) Mouse Hearts

Imene Tabbi-Anneni 1, Jonathan Buchanan 1, Robert C Cooksey 1, E Dale Abel 1
PMCID: PMC2488224  PMID: 18450963

Abstract

The goal of this study was to determine whether inhibiting the renin-angiotensin system would restore insulin signaling and normalize substrate use in hearts from obese ob/ob mice. Mice were treated for 4 wk with Captopril (4 mg/kg·d). Circulating levels of free fatty acids, triglycerides, and insulin were measured and glucose tolerance tests performed. Rates of palmitate oxidation and glycolysis, oxygen consumption, and cardiac power were determined in isolated working hearts in the presence and absence of insulin, along with levels of phosphorylation of Akt and AMP-activated protein kinase (AMPK). Captopril treatment did not correct the hyperinsulinemia or impaired glucose tolerance in ob/ob mice. Rates of fatty acid oxidation were increased and glycolysis decreased in ob/ob hearts, and insulin did not modulate substrate use in hearts of ob/ob mice and did not increase Akt phosphorylation. Captopril restored the ability of insulin to regulate fatty acid oxidation and glycolysis in hearts of ob/ob mice, possibly by increasing Akt phosphorylation. Moreover, AMPK phosphorylation, which was increased in hearts of ob/ob mice, was normalized by Captopril treatment, suggesting that in addition to restoring insulin sensitivity, Captopril treatment improved myocardial energetics. Thus, angiotensin-converting enzyme inhibitors restore the responsiveness of ob/ob mouse hearts to insulin and normalizes AMPK activity independently of effects on systemic metabolic homeostasis.

OBESITY AND DIABETES are important risk factors for the development of cardiovascular disease (1), which is the leading cause of mortality in these individuals (2,3). Insulin resistance is a hallmark of obesity and diabetes and is associated with increased activation of the renin-angiotensin system (RAS), which may contribute to cardiac dysfunction (4,5). Folli et al. (6) have shown that activating the RAS in vascular smooth muscle cells with angiotensin II (Ang II) inhibits insulin signaling via mechanisms that are dependent on Ang II signaling, via Ang II type 1 (AT1) receptors. More recently, Carvalheira et al. (7) reported that Ang II infusion in rats inhibited insulin-mediated activation of phosphatidyl inositide-3-kinase and Akt in the heart. Moreover, in hearts of obese Zucker rats, the expression of Ang II was increased and the associated impairment in insulin signaling was partially restored by the AT1 antagonist, losartan. A metaanalysis of five large clinical studies using angiotensin-converting enzyme inhibitors (ACEIs) in patients with hypertension have shown an approximate 22% reduction in the incidence of type 2 diabetes in these individuals (8). Thus, close interactions exist between insulin resistance in target tissues and activation of the RAS.

Cardiac function and substrate metabolism are altered in obesity and insulin-resistant states and is characterized by increased myocardial fatty acid (FA) oxidation, increased myocardial oxygen consumption (MVO2), and reduced cardiac efficiency (9,10,11,12,13). We and others have also shown that myocardial insulin sensitivity is also impaired (10,14,15,16,17,18). The mechanisms that lead to altered myocardial function and metabolism in obesity are complex and multifactorial but involve increased delivery of FA to the heart, increased activation of FA-regulated transcriptional regulators such as peroxisome proliferator-activated receptor-α, and FA-induced mitochondrial uncoupling (11,19). The contribution of impaired myocardial insulin signaling to the cardiac phenotypes of obesity is less well understood, as therapeutic strategies that increase insulin sensitivity also change the delivery of FA substrates to the heart. Given the potential contribution of increased RAS activation to myocardial insulin resistance, the present study was designed to test the hypothesis that antagonizing the RAS in a mouse model of obesity and insulin resistance would increase myocardial insulin signaling and normalize cardiac substrate metabolism.

In this study we demonstrate that treatment of ob/ob mice with Captopril restored myocardial insulin sensitivity, normalized FA oxidation in the presence of insulin, and reduced the activation of AMP-activated protein kinase (AMPK) without normalizing glucose tolerance or the circulating concentrations of insulin, triglycerides, or free fatty acids.

Materials and Methods

Animals and treatment

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication no. 85-23 revised 1996) and was approved by the Institutional Animal Care and Use Committee of the University of Utah. Homozygous male mice C57BL/6J-lepob (ob/ob) and their respective wild-type controls (C57BL/6J) were obtained at 4 wk of age from the Jackson Laboratory (Bar Harbor, ME). Mice were maintained on a 12-h light, 12-h dark cycle at a room temperature of 21–23 C and had free access to standard chow and water. After 3 d of acclimation, mice were randomly assigned to Captopril treatment or no treatment (Sigma, St. Louis, MO). Treated mice received 4 mg/kg·d Captopril, dissolved in sterile-filtered double-distilled H2O for 4 wk. Captopril concentrations were adjusted during the 4-wk treatment by measuring weekly water consumption and body weight changes for each mouse to maintain an average dosage of 4 mg/kg·d. The control C57BL6/J animals consumed 5.5 ml H2O per mouse per week. Ob/ob mice consumed an average of 7.3 ml H2O per mouse per week (range 6.7 ml/wk at wk 1–8.4 ml per week at the end of the 4 wk treatment period). The chosen Captopril dose is in the range of the effective dose used in humans for treatment of hypertension (8) and in several animal studies (20,21,22).

Measurement of plasma Ang II

Blood was rapidly withdrawn into chilled heparinized tubes containing 150 μl of the following buffer (in moles per liter): pepstatin 2.9 × 10−4; EDTA 9.7 × 10−2; o-phenanthrolene 3 × 10−2, and 0.5 mg/ml enalaprilat. This buffer was used to prevent Ang II breakdown during sampling. Plasma was immediately separated by centrifugation (950 × g for 10 min at 4 C) and stored at −20 C until assayed. Ang II was measured using an Ang II RIA (KMI Diagnostics, Minneapolis, MN).

Glucose tolerance test and determination of serum levels of insulin, free fatty acids (FFAs), and triglycerides (TGs)

Glucose tolerance tests (GTT) were performed after a 6-h fast. A glucose bolus was injected by ip injection (1 mg/g body weight), and blood was collected from the tail vein 30, 60, 90, and 120 min after glucose administration. Blood glucose was determined using the glucose oxidase method with one-touch test strips (Lifescan; Johnson & Johnson Co., Milpitas, CA). In a separate cohort of mice, serum insulin, FFA and TG concentrations were determined during peak feeding (0500 h) and 6 h after food removal. Insulin concentrations were determined using the sensitive rat insulin RIA kit (Linco Research Inc., St. Charles, MO). FFA concentrations were determined using the 1/2-micro fatty acid test kit (Roche Diagnostics, Mannheim, Germany), and TG concentrations were determined using the L-type TG H kit (Wako, Richmond, VA).

Substrate metabolism in isolated working mouse hearts

Hearts from untreated and 4-wk Captopril-treated ob/ob and corresponding wild-type controls were prepared and perfused in the working mode as previously described (10,23). Briefly, mice were anesthetized with ip chloral hydrate (∼0.7 mg/g body weight), and the heart was rapidly excised and arrested in ice-cold buffer. Hearts were perfused with 37 C Krebs Henseleit buffer containing (in millimoles per liter): 118.5 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 0.5 EDTA, gassed with 95% O2 and 5% CO2 and supplemented with 0.4 mm Palmitate bound to 3% BSA and 5 mm glucose, in the presence or absence of 1 nm insulin. Glycolytic rate was determined by measuring the amount 3H2O released from the metabolism of exogenous [5-3H] glucose. Palmitate oxidation was determined in separate perfused hearts by measuring the amount of 3H2O released from [9–10-3H] palmitate. Myocardial MVO2 and cardiac power were determined as previously described (10).

Insulin signaling in isolated perfused hearts

The expression of the insulin receptor (IR), as well as the ability of insulin to increase Akt phosphorylation (Thr308) and tyrosine phosphorylation of the IR and insulin receptor substrate (IRS)-1/2 (p-Tyr PY20), and AMPK (Thr172) phosphorylation were determined by Western blot in homogenates from hearts stimulated or not with insulin during a Langendorff mode perfusion with an EDTA-free 37 C Krebs Henseleit buffer, gassed with 95% O2 and 5% CO2, and supplemented with 0.4 mm palmitate bound to 3% BSA and 5 mm glucose. Mice were anesthetized with ip chloral hydrate, (0.7 mg/g body weight) and the heart was rapidly excised and arrested in ice-cold Krebs Henseleit buffer. The aorta was cannulated and retrogradely perfused at a constant pressure (60 mmHg) for 15 min in the presence or absence of 1 nm insulin. The heart was then freeze-clamped and stored at –80 C until use. Heart homogenates were prepared as previously described (11,24). Western blotting was performed on samples containing equal amounts of proteins after separation of the proteins on sodium dodecyl sulfate-polyacrylamide gels (10% acrylamide), and transfer to a polyvinylidene membrane (Millipore, MA). Membranes were incubated for 1 h at room temperature with PBST (PBS with 0.1% Tween) supplemented with 5% nonfat milk to reduce nonspecific binding, then immunoblotted overnight at 4 C with either mouse p-Tyr (PY20), rabbit IR (Santa Cruz, CA), phospho-Akt (Thr308), Akt, phospho-AMPK (Thr172) or AMPK-α antibody (Cell Signaling Technology, Danvers, MA), at a dilution of 1:1000. Proteins were visualized after incubation of these membranes with secondary goat antirabbit or antimouse HRP-conjugated antibody and detection with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Rockford, IL). Coomassie blue staining R-250 was used to evaluate for protein loading.

Statistical analysis

Data are expressed as mean ± se. Significance (P < 0.05) was determined by ANOVA followed by a Fisher’s protected least significance difference test. Statistical calculations were performed with the Statview 5.0.1 software package (SAS Institute, Cary NC).

Results

Effect of captopril on circulating levels of Ang II

ob/ob mice and controls (C57BL6) were treated or not for 4 wk with therapeutic doses of captopril (4 mg/kg·d). Circulating levels of Ang II were determined in all groups of mice. Relative to wild-type (wt) mice, mean Ang II levels were significantly increased by 1.5-fold in ob/ob mice (Fig. 1, P = 0.02). Treatment with captopril normalized Ang II concentrations in ob/ob mice. Of interest, captopril treatment did not further lower Ang II concentrations in wt mice.

Figure 1.

Figure 1

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Plasma levels of Ang II in treated and untreated mice. Ang II was measured by RIA in four groups of mice: wt control (wt, n = 6), wt captopril treated (wt+CP, n = 4), ob/ob (ob, n = 6), and ob/ob captopril treated (ob+CP, n = 5) after 4 wk of treatment with captopril (4 mg/kg·d). #, P = 0.02 vs. wt.

Heart, body weight, and systemic metabolic parameters of ob/ob mice treated with captopril

As shown in Table 1 and consistent with previous publications (10), 8-wk-old ob/ob mice were significantly obese, compared with controls (body weight 45.41 ± 1.29 g vs. 24.62 ± 0.40 g, P < 0.0001). Moreover, they showed a significant increase in heart weight (dry heart weight 32.64 ± 1.15 vs. 27.21 ± 1.37 mg, P < 0.01). Treatment of ob/ob mice with captopril had no effect on cardiac hypertrophy or weight gain. Glucose tolerance in ob/ob mice treated with captopril was determined by GTT. At 6 h of fasting (t = 0 min), ob/ob mice tended to be hyperglycemic relative to wt mice as shown in Fig. 2A (177.40 ± 21.31 vs. 118.00 ± 8.1 mg/dl, P = 0.07). Fasting glucose levels remained high in ob/ob mice, even after treatment with captopril (198.38 ± 21.68 mg/dl, P = 0.02 vs. wt). Impaired glucose tolerance was not changed in captopril-treated ob/ob mice. Figure 2.B shows that ob/ob mice had elevated insulin levels, compared with wt mice under fasting conditions (0.456 ± 0.05 vs. 0.038 ± 0.01 nm, P < 0.05) and were even more hyperinsulinemic after feeding (2.151 ± 0.165 nm). A similar pattern remained after treatment with captopril indicating that captopril had no effect on hyperinsulinemia in ob/ob mice.

Table 1.

Body and heart weights

BW (g) DHW (mg)
wt 24.62 ± 0.40 (14) 27.21 ± 1.37 (14)
wt+C 23.54 ± 0.37 (15) 27.91 ± 1.01 (15)
ob 45.41 ± 1.29 (15)a 32.64 ± 1.15 (15)a
ob+C 48.60 ± 0.84 (13)a 33.17 ± 1.54 (13)a
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Body (BW) and dry heart weight (DHW) were determined in wt, ob/ob (ob), captopril-treated wild type mice (wt+C) and captopril-treated ob/ob (ob+C) mice. Number of animals per group is in parentheses. 

a

P < 0.05 vs. wt. 

Figure 2.

Figure 2

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Glucose and insulin concentrations in treated and untreated mice. A, GTTs were performed in wt (n = 9), wt captopril (wt+C, n = 10), ob/ob (ob, n = 10), and ob+C (n = 10) mice after 4 wk of treatment with or without captopril (4 mg/kg·d). *, P < 0.05 vs. wt at the same time point. B, Insulin concentrations were determined at both the fasting (fast) and peak feeding (fed) state in wt and ob/ob mice treated or not for 4 wk with captopril (n = 3 per group). *, P < 0.05 vs. wt fast; **, P < 0.05 vs. ob fast; §, P < 0.05 vs. wt+C fast; ¶, P < 0.05 vs. ob+C fast.

Fed and fasting concentrations of FFAs in ob/ob and wt mice treated or not with captopril are shown in Table 2. FFA levels were not increased in the fed and fasted states in ob/ob mice and were not altered by captopril. TG concentrations increased by 63% in wt mice in the fed state when compared with the fasted state (P = 0.02). Similar results were obtained in ob/ob mice indicating that 8-wk-old ob/ob mice in this study were not hypertriglyceridemic and that captopril had no effect on TG levels.

Table 2.

Serum metabolites

wt ob wt+C ob+C
FFAs Fast 1.106 ± 0.055 0.878 ± 0.328 1.210 ± 0.170 1.090 ± 0.079
Fed 1.179 ± 0.211 0.996 ± 0.204 0.667 ± 0.150 1.274 ± 0.081
TGs Fast 1.887 ± 0.264 1.484 ± 0.206 1.512 ± 0.272 1.207 ± 0.047
Fed 3.094 ± 0.781a 3.264 ± 0.410a 2.758 ± 0.051a 3.264 ± 0.251a
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Serum FFAs and TGs were determined after 6 h fasting (fast) and peak feeding (fed) states in wt and ob/ob mice treated or not for 4 wk with captopril (n = 3 per group). 

a

P < 0.05 vs. fast from same group. 

Substrate metabolism in isolated working hearts of ob/ob mice treated with captopril

Glycolysis and fatty acid oxidation were determined in hearts of ob/ob and wt mice treated or not with captopril. Hearts were perfused in the absence or presence of insulin. Insulin increased glycolytic rates by 36% in hearts of wt mice (P < 0.01 vs. wt basal, Fig. 3A). Hearts of ob/ob mice had lower basal glycolytic rates, compared with wt mice (−47% vs. wt, P < 0.0001). As expected, there was no increase in glycolysis in hearts of ob/ob mice in response to insulin, indicating severe cardiac insulin resistance. Hearts of wt mice treated with captopril showed similar glycolytic rates as nontreated wt hearts, indicating that in wt mice, captopril did not affect glycolytic rates. Interestingly, in hearts of ob/ob mice treated with captopril, although basal glycolytic rates remained as low as in nontreated ob/ob mice, glycolysis was increased by 99% in response to insulin stimulation (P < 0.01). Insulin-stimulated glycolytic rates in ob/ob hearts did not reach the levels that were observed in wt mice, but the increase was still highly significant when compared with rates of glycolysis in nontreated ob/ob mice (P < 0.05). These results indicate that captopril improved the ability of insulin to stimulate glycolysis.

Figure 3.

Figure 3

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Substrate metabolism in isolated working hearts. Rates of glycolysis (A) and palmitate oxidation (B) were measured in hearts from wt and ob/ob mice treated or not for 4 wk with captopril (n = 3–5/group). Perfusions were performed at 0.4 mm palmitate and 5 mm glucose in the absence or presence of 1 nm insulin. *, P < 0.05 vs. wt (no insulin); **, P < 0.05 vs. equivalently treated wt; §, P < 0.05 vs. ob/ob captopril treated (ob+C basal). g dwt, gram dry heart weight.

Rates of palmitate oxidation are shown in Fig. 3B. Insulin decreased palmitate oxidation in wt hearts by 37% (P = 0.01). In ob/ob mice, palmitate oxidation rates under basal conditions were 66% higher than in wt mice (P < 0.0001) and, similar to previous reports (10), remained elevated in insulin-perfused hearts, consistent with severe cardiac insulin resistance. Captopril treatment did not affect palmitate oxidation rates in wt hearts. However, in hearts of ob/ob mice treated with captopril, although basal rates of palmitate oxidation remained elevated, the ability of insulin to reduce palmitate oxidation was completely restored, bringing palmitate oxidation rates to levels obtained in insulin-perfused hearts from wt mice.

Oxygen consumption and cardiac performance

As shown in Fig. 4A, insulin significantly reduced cardiac MVO2 by 21% in wt mice (P < 0.05). MVO2 was increased by 28% in hearts of ob/ob mice relative to wt (P < 0.01) and remained high, even in the presence of insulin. This higher oxygen consumption was associated with a higher rate of palmitate oxidation. Hearts of wt mice treated with captopril showed similar results as nontreated controls. Although hearts of ob/ob mice treated with captopril had a similar MVO2 as nontreated ob/ob mice in the absence of insulin, MVO2 was reduced by 13% in response to insulin (P = 0.06). Insulin significantly increased cardiac power in wt mice, but this effect was absent in hearts of ob/ob mice. However, when ob/ob mice were treated with captopril, this effect was partially but significantly restored, as evidenced by a 19% increase in cardiac power relative to captopril-treated ob/ob hearts perfused in the absence of insulin (P < 0.05, Fig. 4B). In all cases, the insulin-mediated increase in cardiac power was attributable primarily to an increase in left ventricular developed pressure, which was actually greatest in ob/ob hearts (supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

Figure 4.

Figure 4

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MVO2 and cardiac power in isolated working hearts. A, The hearts used for MVO2 calculations were the same as those used for palmitate metabolism measurements in Fig. 3 (n = 3–5/group). B, For each perfusion condition, cardiac power data from glycolysis and palmitate oxidation experiments were combined (n = 6–10/group. *, P < 0.05 vs. wt no insulin; **, P < 0.05 vs. equivalently treated wt; ¶, P = 0.06 vs. ob/ob captopril (ob+C basal); §, P < 0.05 vs. ob+C basal.

Insulin and AMPK signaling in the hearts of captopril-treated ob/ob mice

To elucidate signaling mechanisms by which captopril may improve insulin sensitivity in the hearts of ob/ob mice, we measured Akt phosphorylation in ob/ob and wt mouse hearts treated or not with captopril and perfused with or without insulin. Western blot analysis of phospho-Akt (Thr308) normalized to total Akt, is shown in Fig. 5A. As expected, insulin significantly increased Akt phosphorylation in hearts of wt mice, and this effect was severely attenuated in ob/ob mice. Interestingly, treatment with captopril restored the ability of insulin to increase Akt phosphorylation in ob/ob mice. This finding is in accordance with the metabolic changes obtained in the hearts of ob/ob mice and confirms that captopril increases insulin sensitivity in the hearts of these mice. The cardiac expression of the IR was unchanged in all groups of mice (Fig. 5B); however, tyrosine phosphorylation (p-Tyr) of a 96-kDa band, which corresponds to the size of the IR protein was increased in wt mouse hearts in response to insulin stimulation (Fig. 5C). A similar trend was observed for tyrosine phosphorylation of the 180-kDa protein, which likely corresponds to IRS1/2. No significant changes were observed in untreated ob/ob mice hearts, even after stimulation with insulin. Interestingly, captopril induced a significant increase (P < 0.05) in p-Tyr of both 96- and 180-kDa bands in noninsulin-treated ob/ob mouse hearts relative to noninsulin stimulated wild-type mouse hearts, which did not increase further after insulin stimulation. Thus, captopril might increase insulin sensitivity in ob/ob mouse hearts in part by enhancing basal levels of tyrosine phosphorylation of the IR and IRS1/2.

Figure 5.

Figure 5

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Akt and AMPK phosphorylation in isolated perfused hearts. Hearts from wt and ob/ob mice (ob) treated or not for 4 wk with captopril (CP) were perfused in the Langendorff mode in the absence or presence of 1 nm insulin (Ins; n = 3–7/group). A, Phospho-Akt (Thr308) normalized to total Akt (Tot-Akt). B, Total IR normalized to total proteins at 96 kDa. C, p-Tyr (PY20) immunoblot; arrows denote the band corresponding to the IR (96 kDa) and IRS1 (180 kDa) proteins, respectively. Upper panel, Coomassie blue staining (CB) was used to normalize for protein loading. D, Phospho-AMPK (Thr172) normalized to total AMPK were determined in heart homogenates by Western blot. Experiments were repeated twice for each protein, and similar results were obtained. Because no significant changes were observed in phospho-AMPK (Thr172)/total AMPK between basal and insulin stimulated hearts for each respective group of mice, data were pooled. A representative Western blot and a histogram showing densitometry (mean ± se) are shown. *, P < 0.05 vs. wt no insulin; §, P < 0.05 vs. ob+C basal; ***, P < 0.01 vs. ob; $, P < 0.01 vs. ob basal; ##, P < 0.05 vs. ob+ins.

AMPK plays an important role in modulating cardiac energy and ATP availability, and an increase in its phosphorylation may reflect an increase in energetic stress or demand. Because there were no significant changes in AMPK phosphorylation between the insulin stimulated and nonstimulated hearts for each group of mice, data were pooled. Figure 5D shows that AMPK phosphorylation in the heart, expressed as the ratio of phospho-AMPK (Thr172)/total AMPK was increased in ob/ob mice by 84% relative to wt mice (P < 0.001) and was normalized to wt levels after treatment with captopril, suggesting a possible improvement in the energetic status of ob/ob mouse hearts after treatment with captopril.

Discussion

In the present study, we demonstrated that chronic treatment of obese and insulin-resistant ob/ob mice with therapeutic (4 mg/kg) doses of captopril restored insulin sensitivity in the heart as evidenced by the ability of captopril to normalize insulin-mediated suppression of FA oxidation and by restoring the insulin-mediated increase in glycolysis, likely via an increase in insulin-stimulated Akt phosphorylation. These changes occurred without changing basal levels of fatty acid oxidation and glycolysis in isolated hearts and without changing circulating levels of insulin, FFAs, and/or TGs or improving glucose tolerance. There is a large body of evidence to suggest that inhibition of the RAS may improve insulin sensitivity in various models of insulin resistance and obesity, although the results vary, depending on the experimental design, the degree of hyperinsulinemia, and the dose or duration of ACEI treatment (25,26,27,28).

Most animal studies that have investigated the role of captopril in the improvement of whole body insulin sensitivity in obesity and diabetes have used doses of captopril that were 10–50 times higher than the therapeutic doses used in human studies (26,29,30). Carvalho et al. (25), however, showed that acute treatment of 20-wk-old nondiabetic and lean Wistar rats with 2 mg/kg body weight of captopril increased insulin signaling in muscle. We are not aware of any studies that have used these lower doses in animals with insulin resistance and diabetes. Thus, our studies would suggest that at therapeutic doses used to treat various cardiovascular disorders, captopril does not act as a potent insulin sensitizer in peripheral tissues. In contrast, chronic treatment with therapeutic doses of captopril have repeatedly been shown to have beneficial effects on cardiac function, such as preventing the development of congestive heart failure and reducing infarct size in rats after coronary artery ligation (20) or improving cardiac function and reducing cardiac hypertrophy in hypertensive mice (22). However, the consequences of chronic treatment with therapeutic doses of captopril on cardiac metabolism has until now been little investigated. The present study addressed the potential role of chronic treatment with therapeutic doses of captopril on cardiac metabolism in the context of cardiac insulin resistance and obesity. We now show that cardiac insulin sensitivity is enhanced by captopril in the absence of significant changes in indices of whole body insulin sensitivity. These findings suggest that the heart is particularly susceptible to the insulin-sensitizing effects of ACE inhibition and provide new insights into the potential clinical utility of ACE inhibition in reversing functional and metabolic disturbances in the heart that are associated with insulin resistance and obesity.

A 4-wk treatment with captopril (4 mg/kg·d) normalized Ang II levels in ob/ob mice to the level of wild-type mice, indicating therapeutic efficacy. Although blood pressure was not measured in these mice, it has previously been shown that in diabetic rats, a 4-wk treatment with low doses of captopril induced a reduction in blood pressure in these animals (21). Another study using an animal model of cardiac hypertrophy showed that chronic treatment with 4 mg/kg·d of captopril reduced arterial pressure in these mice, whereas no significant changes were observed in captopril-treated wild-type mice (22). This was accompanied by a reduction in plasma levels of Ang II in treated diabetic or hypertensive mice, whereas no changes were noted in wild-type treated mice. Thus, in terms of Ang II levels, the results in the present study in an animal model of obesity are similar to prior studies, in that Ang II levels were normalized in obese animals but were not changed by captopril in lean animals. Because we did not measure blood pressures in these studies, we cannot definitively conclude that the normalization of insulin sensitivity by captopril in ob/ob mice is solely a function of normalization of the elevated levels of Ang II. However, it should be emphasized that we have previously shown that ob/ob mice do not have increased blood pressure relative to control mice at this age (23).

As previously reported by our group and others, insulin resistance in obesity and diabetes is associated with changes in cardiac function and metabolism. An increase in fatty acid availability and increased myocardial fatty acid uptake causes a shift in substrate metabolism, which increases fatty acid oxidation and oxygen consumption and reduces cardiac efficiency (10,11,13,23,31,32). In the present study, hearts of ob/ob mice were profoundly insulin resistant as evidenced by an impaired ability of insulin to reduce palmitate oxidation or to increase glycolysis and increase Akt phosphorylation. Thus, fatty acid oxidation was increased and glycolysis decreased in ob/ob mouse hearts, and insulin failed to modulate cardiac metabolism. These changes in metabolism were paralleled by changes in MVO2 and cardiac power. Insulin treatment of wild-type hearts reduced MVO2 presumably by increasing glucose use and reducing FA use. In insulin-resistant ob/ob mouse hearts, insulin had no effect on MVO2 but captopril treatment led to an insulin-mediated reduction in MVO2, which is consistent with the insulin-mediated reduction in FA use in these hearts. Insulin has a positive inotropic effect in perfused hearts. This was absent in untreated ob/ob mouse hearts and was restored in captopril-treated hearts. The significant improvement in cardiac power was the result of an increase in LV developed pressure (supplemental Table 1), and because hearts of captopril-treated ob/ob mice remained hypertrophied, the increase in cardiac power, which is normalized to cardiac weight, was blunted relative to insulin-perfused control hearts. All of these observations provide convincing evidence that captopril improved insulin sensitivity of ob/ob mouse hearts.

It is noteworthy that captopril treatment did not alter substrate metabolism in hearts that were perfused in the absence of insulin, indicating that captopril did not influence the underlying defects in substrate metabolism in ob/ob mouse hearts and that its effect was mainly due to changes in insulin responsiveness. However, given that captopril-treated ob/ob mice remain hyperinsulinemic in vivo, the fact that myocardial insulin-responsiveness was restored raises the possibility that the elevated in vivo concentrations of insulin in captopril-treated ob/ob mice could promote increased glucose use, decreased FA use, and MVO2 in vivo, thereby increasing cardiac efficiency. Moreover, the increase in insulin sensitivity might render these hearts more likely to benefit from the cardioprotective effects of insulin, particularly in the context of myocardial ischemia (33). The lack of an effect of captopril to modulate rates of fatty acid oxidation or glycolysis in the absence of insulin differs from recently published data obtained in hearts of streptozotocin (STZ) diabetic rats, in which chronic treatment with captopril was shown to improve glucose metabolism in isolated perfused Langendorff hearts by modulating fuel metabolic gene expression in the heart (21). It is difficult to directly compare the heart perfusion data in these two studies because hearts in the study of STZ diabetic rats were perfused with glucose in the absence of palmitate, which if present might have influenced myocardial glucose use in diabetic hearts. Second, the underlying pathophysiology in ob/ob mice, i.e. insulin resistance and obesity, with impaired glucose tolerance is likely to be distinct from severely hyperglycemic and insulin-deficient STZ diabetic animals. Nevertheless, the studies are similar in that the improvement in cardiac metabolism occurred in the absence of any changes in systemic glucose or lipid homeostasis. In our study, this effect also occurred in the absence of any change in weight gain in ob/ob mice, suggesting that adipose tissue mass was most likely unaffected by the treatment.

These findings suggest that the improved insulin sensitivity in the hearts of ob/ob mice may be the result of a direct effect of captopril on the myocardium, acting most likely via modulation of insulin signaling. This was supported by the restoration of the ability of insulin to increase (Thr308) Akt phosphorylation in the hearts of captopril-treated ob/ob mice, thereby reversing the severe signaling defect that was present in hearts of untreated ob/ob mice. Captopril also increased tyrosine phosphorylation of 96- and 180-kDa proteins in hearts of ob/ob mice, consistent with an increase in both IR and IRS1/2 phosphorylation, thereby suggesting that captopril might promote myocardial insulin sensitivity by enhancing proximal steps in the insulin signaling pathway independently of changes in protein levels of the IR.

The improvement in insulin sensitivity of ob/ob mouse hearts occurred in the absence of any evidence of increased peripheral insulin sensitivity or changes in coronary flow (supplemental Table 1). There are a number of potential mechanisms by which ACE inhibition could increase myocardial insulin sensitivity. In smooth muscle cells, Ang II may inhibit the insulin signaling pathway by increasing the serine phosphorylation of IRS1 and reducing its association with phosphatidyl inositide-3-kinase (6). In addition, in obese Zucker rats, cardiomyocyte expression of Ang II was increased, and this was associated with reduced insulin-mediated tyrosine phosphorylation of IRS1 and impaired insulin signaling (7). This defect was partially restored by the AT1 receptor antagonist, losartan. Protein kinase C is an important intracellular mediator of Ang II action via activation of AT1 receptors. Treatment of STZ diabetic rats with protein kinase C inhibitors was shown to improve cardiac metabolism and prevented the inhibition of insulin-stimulated glucose uptake in cardiomyocytes by exogenous fatty acids (21). Moreover, evidence exists that captopril and other ACEIs may have antioxidant properties, and recent studies have suggested that oxidative stress might be an important underlying mechanism in the pathogenesis of insulin resistance (34,35,36).

Dyntar et al. (37) showed that exposure of adult rat cardiomyocytes to palmitate increased apoptosis by activating the mitochondrial apoptotic pathway, and some studies suggest that ACEI may have antiapoptotic effects in cardiomyocytes (38,39). In our study, captopril reduced palmitate oxidation in ob/ob mouse hearts in response to insulin stimulation, raising the possibility that the reduction in FA-oxidation rates in insulin-perfused hearts from captopril-treated ob/ob mice could reflect a reduction in myocardial FA use, which might limit lipotoxicity. However, we have not measured the impact of captopril on myocardial lipid accumulation or apoptosis, which is a topic for further study. Thus, there are multiple mechanisms that might contribute to the beneficial impact of ACE inhibition on myocardial insulin sensitivity in ob/ob mice. Importantly we now show that by increasing insulin signaling in the heart of obese and insulin-resistant mice, metabolic flexibility can be restored and patterns of substrate use in the presence of insulin can be normalized.

We have previously shown that in addition to insulin resistance, mitochondrial dysfunction may contribute to contractile dysfunction in the heart in obesity (11,40). AMPK is an important sensor of cellular energy reserves. Thus, it was not unexpected that AMPK activity as assessed by the phosphorylation state of AMPK was increased in ob/ob mouse hearts. Kewalramani et al. (41) also showed that in rats with acute diabetes, AMPK phosphorylation was increased in the heart. Our findings differ from those reported by Wang and Unger (42) in which AMPK phosphorylation was significantly reduced in hearts of both Zucker diabetic rats (ZDF fa/fa) and 10- to 15-wk-old ob/ob mice. An important difference between our study and that of Wang and Unger is that in our study AMPK activity was evaluated in hearts that were perfused for 30 min, whereas in the study by Wang and Unger, hearts were removed immediately from euthanized mice. The isolation of and preparation of hearts before perfusion is associated with a brief period of ischemia that could activate AMPK. Thus, the increase in AMPK phosphorylation in hearts from untreated ob/ob mice could indicate reduced energetic reserves in ob/ob hearts, which is consistent with our previously published data showing mitochondrial dysfunction in ob/ob hearts (11,40). Captopril treatment normalized AMPK activity in perfused ob/ob mouse hearts. These changes are consistent with the hypothesis that in addition to enhancing insulin sensitivity, captopril treatment might also act to increase myocardial energetics.

In conclusion, this study has demonstrated that a 4-wk treatment with therapeutic doses of captopril completely restored the ability of insulin to modulate fatty acid oxidation and improved its ability to increase glycolysis by increasing insulin signaling in the heart, without significant effects on systemic metabolic homeostasis. These changes in cardiac metabolism were associated with a reduction in MVO2 and an improvement in cardiac performance in the presence of insulin. Moreover, captopril normalized AMPK activity in hearts of ob/ob mice, suggesting an improvement of the energetic status in the heart of obese insulin-resistant mice. These findings provide new insights into potential beneficial effects of ACE inhibition in the treatment of obesity and diabetes-related myocardial dysfunction.

Supplementary Material

[Supplemental Data]
en.2007-1646_index.html (756B, html)

Footnotes

This work was supported by National Institutes of Health Grant RO1 HL73167 (to E.D.A., who is an established investigator of the American Heart Association).

Disclosure Statement: The authors have no conflicts of interest to report.

First Published Online May 1, 2008

Abbreviations: ACEI, Angiotensin-converting enzyme inhibitor; AMPK, adenosine monophosphate activated protein kinase; Ang II, angiotensin II; AT1, Ang II type 1; FA, fatty acid; FFA, free fatty acid; GTT, glucose tolerance test; IR, insulin receptor; IRS, insulin receptor substrate; MVO2, myocardial oxygen consumption; p-Tyr, phosphotyrosine; RAS, renin angiotensin system; STZ, streptozotocin; TG, triglyceride; wt, wild type.

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[Supplemental Data]
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