Metabolic gene switching in the murine female heart parallels enhanced mitochondrial respiratory function in response to oxidative stress M. Faadiel Essop 1,2 , W. Y. A. Chan 2 and Heinrich Taegtmeyer 3 1 Department of Physiological Sciences, Stellenbosch University, South Africa 2 Hatter Heart Research Institute, Faculty of Health Sciences, University of Cape Town, South Africa 3 Department of Internal Medicine, Division of Cardiology, University of Texas, Houston Medical School, TX, USA Premenopausal women have a lower risk for develop- ing cardiovascular disease as compared to age-matched males [1]. Moreover, experimental studies show increa- sed resistance to ischemia⁄ reperfusion injury in female versus male hearts [2,3]. The molecular regulatory mechanisms underlying such gender-based differences are unclear. However, estrogen may play a key role in this process [4], and is thought to signal its cardio- protective effects via the prosurvival serine-threonine protein kinase, Akt (also known as protein kinase B) [2]. In agreement with this, elevated levels of activated Akt in female hearts are linked to improved cardiac cell survival [5], and a recent study implicated the PI3-K ⁄ Akt signaling pathway in estrogen-mediated cardioprotection [6]. Adaptive metabolic remodeling is considered to be an important component of cardioprotective mecha- nisms in response to decreased oxygen supply. For example, enhanced glucose utilization is proposed to confer cardioprotective effects in response to ische- mia ⁄ reperfusion [7]. Conversely, higher rates of fatty acid oxidation during ischemia may uncouple mito- chondrial oxidative phosphorylation and ⁄ or increase proton production, contributing to impaired contractile Keywords bioenergetics; cardiovascular disease; gender differences; gene expression; mitochondrial respiration Correspondence M. F. Essop, Department of Physiological Sciences, Stellenbosch University, Room 2009, Mike De Vries Building, Merriman Avenue, Stellenbosch 7600, South Africa Fax: +27 21 808 3145 Tel: +27 21 808 4507 E-mail: mfessop@sun.ac.za (Received 14 December 2006, revised 14 August 2007, accepted 17 August 2007) doi:10.1111/j.1742-4658.2007.06051.x The mechanisms underlying increased cardioprotection in younger female mice are unclear. We hypothesized that serine-threonine protein kinase (protein kinase B; Akt) triggers a metabolic gene switch (decreased fatty acids, increased glucose) in female hearts to enhance mitochondrial bio- energetic capacity, conferring protection against oxidative stress. Here, we employed male and female control (db ⁄ +) and obese (db ⁄ db) mice. We found diminished transcript levels of peroxisome proliferator-activated receptor-alpha, muscle-type carnitine palmitoyltransferase 1 and pyruvate dehydrogenase kinase 4 in female control hearts versus male hearts. More- over, females displayed improved recovery of cardiac mitochondrial respi- ratory function and higher ATP levels versus males in response to acute oxygen deprivation. All these changes were reversed in female db ⁄ db hearts. However, we found no significant gender-based differences in levels of Akt, suggesting that Akt-independent signaling mechanisms are respon- sible for the resilient mitochondrial phenotype observed in female mouse hearts. As glucose is a more energetically efficient fuel substrate when oxy- gen is limiting, this gene program may be a crucial component that enhances tolerance to oxygen deprivation in female hearts. Abbreviations Akt, serine-threonine protein kinase (protein kinase B); GLUT, glucose transporter; MCAD, medium-chain acyl-CoA dehydrogenase; mCPT1, muscle-type carnitine palmitoyltransferase 1; PDK-4, pyruvate dehydrogenase kinase 4; PGC-1, peroxisome proliferator-activated receptor- gamma coactivator-1; PPARa, peroxisome proliferator-activated receptor-alpha; UCP3, uncoupling protein 3. 5278 FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS function [8]. Likewise, high fatty acid oxidation rates in the diabetic heart results in reduced cardiac effi- ciency [9]. In addition to its cytoplasmic role, Akt can also translocate to the nucleus, where it has transcriptional effects. For example, constitutively activated Akt resulted in reduced myocardial gene expression of peroxisome proliferator-activated receptor-alpha (PPARa) and peroxisome proliferator-activated recep- tor-gamma coactivator-1 (PGC-1), pivotal nuclear regulators of numerous fatty acid metabolic genes [10]. In light of this, we hypothesized that Akt trig- gers a metabolic gene switch from fatty acids to increased glucose metabolism in female hearts, thereby conferring protection against acute oxygen deprivation. Moreover, we propose that this selective advantage would be lost with the onset of obesity and type 2 diabetes. Results and Discussion The main finding of this study is the identification of a metabolic gene switch from fatty acids to glucose in the murine female heart at baseline linked to enhanced mitochondrial respiratory capacity in response to oxi- dative stress. Moreover, we found that this ‘female advantage’ is lost with the onset of obesity ⁄ type 2 dia- betes. However, our data show that these changes occurred in an Akt-independent manner. Laboratory-based studies show that female hearts exhibit increased resilience to ischemia ⁄ reperfusion injury and cell death as compared to males [2,3]. Estrogen is thought to play an important role in this process, and is proposed to signal its cardioprotection via the prosurvival kinase Akt [2,4]. For example, higher levels of activated Akt were reported in pre- menopausal women as compared to men [11]. More- over, estrogen stimulation resulted in elevated nuclear levels of activated Akt [11]. We tested our hypothesis in male and female hearts collected from db ⁄ db mice, a well-characterized model of obesity-induced type 2 diabetes (Table 1). However, we did not find any sig- nificant changes in Akt activation in female hearts at baseline as compared to males (Fig. 1). To further investigate our hypothesis, we measured steady-state transcript levels of various cardiac meta- bolic genes. Here, myocardial PPARa gene expression was reduced in females at baseline (P<0.001 versus male controls) (Fig. 2A). In parallel, expression of the genes encoding muscle-type carnitine palmitoyltransfer- ase 1 (mCPT1) and medium-chain acyl-CoA dehydro- genase (MCAD) (both PPARa target genes) was Table 1. Baseline characterization of male and female obese mice. Values are expressed for 18–20-week-old male and female db ⁄ + versus db ⁄ db mice (mean ± SEM, n ¼ 10 animals). ** P < 0.001 compared with age-matched db ⁄ +mice. Body weight (g) Heart ⁄ body weight ratio (· 1000) Fasting blood glucose (mmolÆL )1 ) Male db ⁄ + 28.0 ± 0.7 4.0 ± 0.1 5.5 ± 0.4 db ⁄ db 47.2 ± 1.7** 2.1 ± 0.1** 26.8 ± 0.9** Female db ⁄ + 20.8 ± 0.5 3.6 ± 0.1 4.2 ± 0.2 db ⁄ db 44.7 ± 1.2** 2.2 ± 0.1** 25.9 ± 1.2** A B C D Fig. 1. Immunohistochemical analysis of phospho-Akt in male and female control (db ⁄ +) versus obese (db ⁄ db) mice. (A) Male control, (B) male obese, (C) female control and (D) female obese mice. Phospho-Akt is stained brown and the nucleus is counter- stained blue. Magnification · 200 (n ¼ 6). M. F. Essop et al. Metabolic gene switching in murine female heart FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS 5279 diminished in female hearts (Fig. 2B,C). However, uncoupling protein 3 (UCP3) expression remained unaltered versus males (Fig. 2D). Pyruvate dehydroge- nase kinase 4 (PDK-4) transcript levels were markedly attenuated in female hearts at baseline (P<0.001 ver- sus male controls) (Fig. 2E), whereas glucose trans- porter (GLUT) 4 expression was not significantly different as compared to males (Fig. 2F). Interestingly, we found that gene changes in the female heart were abolished with the onset of obesity ⁄ type 2 diabetes. Together, these data suggest that murine female hearts, unlike male hearts, may display a reduced reliance on fatty acids as fuel substrate. In agreement, lower PDK-4 gene expression in female hearts indicates that myocardial glucose metabolism may be increased in parallel. As optimization of glucose metabolism is increasingly highlighted as a therapeutic intervention for ischemia-induced and ischemia–reperfusion-induced cardiac damage [7], our data provide a novel mecha- nism for how signaling cascades induce transcriptional pathways to augment glucose-mediated cardioprotec- tion in younger females. In agreement with this con- cept, we propose that this advantage is eliminated in obesity ⁄ type 2 diabetes, due to higher fatty acid utili- zation by the diabetic heart [9]. To functionally assess the significance of these findings, we evaluated mitochondrial respiratory function at baseline and in response to acute oxygen AB CD EF Fig. 2. Cardiac metabolic gene expression in male and female control versus obese mice. (A) PPARa, (B) mCPT1, (C) MCAD, (D) UCP3, (E) GLUT4 and (F) PDK-4 in male and female obese versus control mice. Data are expressed as mean ± SEM. *P < 0.001 versus male control mice; **P < 0.01 versus male obese mice; # P < 0.01 versus female control mice; ## P < 0.001 versus female control mice; § P < 0.001 versus male control mice. Metabolic gene switching in murine female heart M. F. Essop et al. 5280 FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS deprivation. Here, females displayed increased mito- chondrial respiratory function at baseline as compared to males (Table 2). The efficiency of respiration (ADP ⁄ O) and ADP phosphorylation rate were similar between male and female mitochondria at baseline. In contrast to what was expected, our data suggest that male obese mice coped well when challenged by oxi- dant stress; that is, respiratory function and myo- cardial ATP levels were not significantly altered (Fig. 3A,B). We are unsure why this occurred, and propose that the stress applied was not severe enough or that some adaptive mechanisms were initi- ated in the male obese mice. However, female controls exhibited enhanced recovery of state 3 mitochondrial respiration versus males in response to oxygen lack (23.6 ± 1.6 versus 16.4 ± 2.6 nmolÆmin )1 Æmg )1 pro- tein) (P < 0.05) (Fig. 3A). Moreover, this was associ- ated with higher postanoxic myocardial mitochondrial ATP levels in females as compared to male controls (39.0 ± 2.7 versus 26.2 ± 2.6 lmolÆmg )1 protein) (P<0.05) (Fig. 3B). Again, these changes were abol- ished in female db ⁄ db heart mitochondria. Together, these data suggest that the greater reliance of female murine cardiac mitochondria on glucose than on fatty acids as compared to their male counterparts may result in enhanced cardioprotection when they are challenged by a biological stress, e.g. oxygen lack. Enhanced glucose utilization in response to oxygen deprivation may occur because glucose is a more oxy- gen-efficient fuel substrate for the generation of ATP as compared to fatty acids. Also, fatty acid-mediated uncoupling of mitochondrial oxidative phosphoryla- tion may result in diminished mitochondrial ATP pro- duction for a given rate of oxygen consumption [12]. However, it is likely that additional mechanisms play a role, as increased myocardial ATP levels were observed in female controls in response to oxygen stress. We propose that estrogen-mediated mitochondrial biogene- sis may also be implicated in this process. In agree- ment, recent studies reported that the orphan nuclear receptor estrogen receptor-alpha, proposed to mediate estrogen signaling, may play a transcriptional role in mitochondrial biogenesis [13,14]. Further studies are, however, required to investigate this possibility. Limitations Although the gene data in this study support a fuel substrate switch away from fatty acids, further studies measuring actual cardiac fuel substrate utilization are Table 2. Cardiac mitochondrial respiration for male and female obese mice. Heart mitochondria were isolated from 18–20-week-old mice as described. Values are expressed as mean ± SEM (n ¼ 7 animals). * P < 0.05 compared with male db ⁄ +mice. ** P < 0.05 compared with male obese db ⁄ db mice. Male Female db ⁄ +db⁄ db db ⁄ +db⁄ db State 2 respiration (nmolÆmin )1 Æmg )1 protein) 29.4 ± 1.6 32.6 ± 1.9 36.6 ± 1.7* 35.0 ± 2.0 State 3 respiration (nmolÆmin )1 Æmg )1 protein) 145.0 ± 9.6 165.8 ± 9.8 175.6 ± 7.1 177.8 ± 12.9 State 4 respiration (nmolÆmin )1 Æmg )1 protein) 33.5 ± 2.0 30.7 ± 2.4 38.8 ± 1.03 36.8 ± 1.6 ADP ⁄ O 2.5 ± 0.1 2.6 ± 0.1 2.2 ± 0.04 2.3 ± 0.1** Phosphorylation rate (nmolÆmin )1 Æmg )1 protein) 363.4 ± 28.3 436.0 ± 32.3 381.0 ± 20.4 411.6 ± 40.0 AB Fig. 3. Improved mitochondrial respiratory function in female control mouse hearts in response to oxygen lack. (A) Percentage recovery of state 3 respiration after 20 min of oxygen deprivation. (B) Total postanoxic mitochondrial ATP levels in male and female cardiac mitochondria. Data are presented as mean ± SEM. *P < 0.05 versus male control mice; # P < 0.05 versus female control mice; ## P < 0.001 versus female control mice (n ¼ 7). M. F. Essop et al. Metabolic gene switching in murine female heart FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS 5281 required to confirm these findings. Moreover, mito- chondrial respiratory functional analyses were only performed using fatty acids as substrate. Additional studies measuring mitochondrial respiratory function using a more representative substrate for glucose oxi- dation should provide additional insights into this interesting question. Conclusions In summary, we have identified a novel metabolic gene switch (decrease in fatty acid utilization, increase in glucose utilization) in the murine female heart. Moreover, our data suggest that these changes occur in an Akt-independent manner. Further studies are therefore required to identify the precise signaling mechanisms that control the metabolic remodeling that we observed in the female murine heart at base- line. As glucose is a more energetically efficient fuel substrate than fatty acids when oxygen is limiting, we believe that this mechanism may represent a crucial component underlying enhanced recovery in younger female hearts in response to oxygen deprivation. Experimental procedures Animals To investigate our hypothesis, we employed 18–20-week- old male and female leptin-receptor deficient (db ⁄ db) (BKS.Cg-m+ ⁄ +Lepr db ⁄ J strain) and heterozygous (db ⁄ +) mice. Mice were obtained from Jackson Laboratory (Bar Harbor, ME) and exposed to a reverse 12 h light ⁄ 12 h dark cycle with free access to standard mouse chow and water. Two weeks before mice were killed, blood glucose levels were measured using a glucose meter (ACCU- CHECK Active Meter; Roche, Basel, Switzerland) after a 6 h fast. All animal experiments were approved by the University of Cape Town’s Animal Research Ethics Committee, and the investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996). Immunohistochemistry Heart tissues were fixed with paraformaldehyde and embed- ded in paraffin wax. Paraffin sections (2–3 lm thick) were dewaxed in xylene and rehydrated through graded ethanol. Expression of phospho-Akt was detected using mouse monoclonal anti-phospho-Akt (Ser473) IgG (Cell Signaling, Danvers, MA). Gene analysis RNA extraction and real-time quantitative RT-PCR of samples were performed using previously described methods [15]. Primers for gene analysis in this study have been described previously [15–18]. We determined transcript levels of: mCPT1, the rate-limiting mitochondrial fatty acid-transferring enzyme; MCAD, a representative fatty acid b-oxidation enzyme; PPARa, a key transcriptional regulator of fatty acid genes; UCP3; the cardiac-enriched GLUT4; and PDK-4, an indirect inhibitor of glucose oxida- tion. Gene expression was normalized to 18S rRNA. Mitochondrial isolation and respiration studies Animals were anesthetized using intraperitoneal sodium pentobarbital (50 mgÆkg )1 ) and heparinized to prevent blood clotting. Mouse hearts were dissected and mitochon- dria isolated as described previously [19], with modifica- tions. Ventricular tissue was homogenized in ice-cold potassium ⁄ EDTA (KE) buffer (0.18 m KCl, 10 m m EDTA, pH 7.4), and the homogenate was centrifuged at 660 g for 5 min at 4 °C in a fixed angle rotor (Sigma 202 MK, Heraues, Germany). The supernatant was again centrifuged at 960 g for 5 min, and the mitochondrial pellet was resus- pended in 50 lL of KE buffer. Mitochondrial respiratory rates were polarographically measured at 25 °Cas described previously [20], with modifications. Isolated mito- chondria were added to the electrode chamber containing incubation medium (25 mm Tris ⁄ HCl, 250 mm sucrose, 8.5 mm KH 2 PO 4 , pH 7.4). We employed a mixture of 5mm malate and 25 lm palmitoyl-l-carnitine as oxidative substrates. State 2 respiration (resting) was measured after addition of oxidative substrates, and state 3 respiration after the addition of 300 lm ADP to the electrode chamber. To test the ability of mitochondria to withstand oxidative stress, 3 mm ADP was added after state 4 respiration, and the chamber was closed and sealed for a 20 min period. As a result, oxygen in the closed chamber would be used to convert ADP to ATP, and an anaerobic condition estab- lished. After 20 min of oxygen lack, the chamber was reoxygenated for 6 min, and the percentage recovery of state 3 respiration was calculated as the ratio of oxygen consumption before and after oxygen lack. All mitochon- drial polarographic studies were normalized to total mito- chondrial protein content [21]. Mitochondrial ATP levels Postanoxic mitochondrial ATP concentration was assayed using a luciferin ⁄ luciferase luminometry luminescence method [22] with modifications. Freshly isolated mitochon- dria were placed in boiling water (3 · sample volume) for Metabolic gene switching in murine female heart M. F. Essop et al. 5282 FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS 10 min, and thereafter on ice for an additional 10 min to disrupt mitochondrial membranes. The mixture was sub- sequently centrifuged at 960 g for 5 min at 4 °C in a fixed angle rotor (Sigma 202 MK). Ten microliters of supernatant was added to 175 lL of distilled water and 25 lL of ATP assay mix [Bioluminescent Somatic Cell Assay Kit (FL-AA); Sigma, St Louis, MO] containing luciferin and luciferase. The reaction of the luciferin ⁄ luciferase mixture, energy donor (ATP) and oxygen results in the emission of light. The intensity of the bioluminescence was detected using a luminometer, and the mitochondrial ATP concentration was determined. Mitochondrial protein concentration was deter- mined [21], and a standard curve was constructed using 1 · 10 )5 m to 1 · 10 )9 m ATP. The concentration of mito- chondrial ATP was expressed as micromoles of ATP per milligram of mitochondrial protein. 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Essop et al. 5284 FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS . Metabolic gene switching in the murine female heart parallels enhanced mitochondrial respiratory function in response to oxidative stress M fatty acids to glucose in the murine female heart at baseline linked to enhanced mitochondrial respiratory capacity in response to oxi- dative stress. Moreover,