In this study, we show that phosphoinositide-3 Kinase (PI3K) is a critical modulator of mitochondrial fatty acid (FA) metabolism in the heart and that this metabolic regulation is independent of Akt. Constitutive activation of PI3K in the heart is sufficient to increase FA utilization and selectively up-regulate mitochondrial oxidative capacity for FA substrates. Inhibition of PI3K prevents the increase in FAO capacity that occurs in response to physiological cardiac hypertrophy, despite increased PGC-1α mRNA and protein levels. Downstream of PI3K, disruption of Akt signaling did not prevent the mitochondrial adaptations to exercise or PI3K activation. Inhibition of cardiac Akt signaling in the context of exercise training or PI3K activation attenuates hypertrophy, but does not diminish mitochondrial respiration or FAO enzyme activity. Conversely, constitutive activation of cardiac Akt results in cardiac hypertrophy but impairs mitochondrial function, thereby dissociating a conserved signal for cellular growth from mitochondrial adaptations. Thus, the current study identifies a necessary role for PI3K in coordinating myocardial fatty acid oxidative capacity with physiological cardiac hypertrophy, independently of PGC-1α expression. We also demonstrate that the long-term metabolic and mitochondrial effects of PI3K can be dissociated from its downstream target Akt, thereby representing a novel paradigm in long-term regulation of metabolism by PI3K signaling.
Previous studies have shown that inhibition of PI3K blocks the heart’s ability to hypertrophy in response to exercise training (McMullen et al., 2003
). We currently show that PI3K inhibition in hearts from exercise-trained mice also prevents the increase in mitochondrial FA oxidative capacity, despite a significant increase in PGC-1α expression. The requirement of p110α in the regulation of cardiac growth and the associated metabolic adaptation implies a tight link between physiological hypertrophy and enhanced mitochondrial FA oxidative capacity. Activation of PI3K therefore leads to a balanced increase in oxidative energy production and cardiac growth that may protect against the progression to heart failure seen in pathological hypertrophy. This protective effect may be specific to the growth factor and exercise training responsive Class Ia PI3-kinases. Indeed, previous studies have shown that whereas class Ia PI3Ks regulate hypertrophy, class Ib PI3Ks modulate contractility in the heart (Crackower et al., 2002
Acute activation of PI3K in the heart, as occurs with insulin stimulation, alters substrate utilization by increasing glucose utilization and reducing FA oxidation (Belke et al., 2002
). PI3K is necessary for insulin-mediated glucose uptake and thereby plays a primary role in acutely increasing glucose utilization and reciprocally decreasing FA oxidation in response to insulin (Pessin and Saltiel, 2000
). Decreased FA utilization induced by insulin stimulation seems at odds with our observation that increased PI3K activation and increased FA oxidation are associated with exercise-induced or physiological cardiac hypertrophy (Burelle et al., 2004
; Luo et al., 2005
; McMullen et al., 2003
). The current study distinguishes between acute and chronic PI3K signaling and confirms the role of chronic PI3K signaling in increasing mitochondrial FA oxidative capacity during physiological cardiac hypertrophy. Thus, while acute activation of PI3K by insulin stimulation increases glucose utilization and suppresses FA oxidation in the heart, chronic activation of PI3K in the heart enhances FA oxidation by increasing mitochondrial oxidative capacity for FA substrates.
Quantification of mRNA levels of FA oxidation enzymes revealed divergent downstream effects of PI3K signaling in the heart. Thus, activation of PI3K enhances FA oxidative capacity in the absence of an increase in transcript levels of FAO enzymes, with the exception of MCAD, thereby suggesting that activation of PI3K in response to physiologic stimuli may enhance mitochondrial function via post-transcriptional mechanisms. In contrast, inhibition of PI3K is associated with decreased mRNA levels of FAO enzymes and prevents their increase in response to exercise training. This suggested that PI3K signaling might modulate expression or activity of the transcriptional regulators of FAO gene expression. However, dnPI3K hearts showed no changes in PPARα, PGC-1β, or ERRα mRNA levels, which are transcriptional regulators of enzymes and subunits in FAO and OXPHOS pathways in the heart (Huss and Kelly, 2004
). Indeed, despite diminished mitochondrial FAO capacity in hearts from exercise-trained and sedentary dnPI3K mice, we observed increased PGC-1α mRNA and protein levels, to a similar degree as observed in exercise-trained wildtype hearts. Thus down-regulation of FAO gene expression in dnPI3K hearts cannot be accounted for by reduced expression of the transcriptional regulators of FAO enzymes. However, the possibility of changes in the activity of PPAR transcription factors or PGC-1 transcriptional co-activators cannot be ruled out. While diminished activity of transcriptional co-activators may account for the down-regulation of FAO transcript levels in the heart when PI3K is inhibited, the lack of a significant increase in mitochondrial FAO genes in caPI3K hearts would argue against a change in the activity of transcriptional regulators of mitochondrial FA oxidative capacity when PI3K is chronically activated in the heart.
Our results show that exercise training synergistically increases mitochondrial FAO, PGC-1α expression, and expression of mitochondrial genes, yet chronic activation of PI3K in the heart does not recapitulate this transcriptional profile. The differences in theses two models may be a consequence of the duration of PI3K activation. Intermittent activation of PI3K, as occurs in exercise training, may be more beneficial for mitochondrial function, and is a potential reason why exercise-trained hearts and hearts with constitutive activation of PI3K differ in the expression of mitochondrial enzymes and their transcriptional regulators. Indeed, we have evidence that models of chronic Akt activation in the heart show diminished mitochondrial function and decreased expression of PGC-1α and its target genes (manuscript in preparation). Thus it appears that intermittent activation of PI3K during exercise training is necessary for coordinated increases in mitochondrial enzyme gene expression and FAO capacity, but constitutive activation of PI3K signaling leads to sustained Akt activation, which can suppress the increased expression of PGC-1α and its target mitochondrial FAO enzymes in the heart. Nevertheless our data strongly suggests that sustained PI3K activation might be sufficient to promote increased mitochondrial FAO independently of a coordinate increase in PGC-1α-mediated transcriptional upregulation.
Our initial efforts to determine if Akt mediates the mitochondrial adaptations in response to PI3K signaling revealed that constitutive activation of Akt in the heart leads to diminished mitochondrial FAO capacity. This result might not be surprising in light of data from many groups showing that chronic Akt activation in the heart is detrimental to cardiac function (Nagoshi et al., 2005
; O’Neill and Abel, 2005
; Shiojima et al., 2005
). In addition, hearts from mice with germline deletion of Akt1 and Akt2 and hearts with reduced Akt signaling via overexpression of a kinase dead Akt1 isoform respond normally by increasing mitochondrial function in response to exercise training. These observations led us to hypothesize that the modulation of mitochondrial function by PI3K is independent of Akt signaling. We confirmed the findings previously reported by Shioi and colleagues that cardiac-restricted expression of a kinase dead Akt1 diminishes signaling downstream of Akt in the heart and attenuates cardiac hypertrophy in caPI3K mice (Shioi et al., 2002
). We further show that inhibition of Akt signaling does not prevent the PI3K-mediated increase in mitochondrial FA oxidative capacity. Additionally, in contrast to dnPI3K hearts in which ATP synthesis was reduced with palmitoyl-carnitine, attenuation of Akt signaling in kdAkt (Shioi et al., 2002
) and DTG hearts increased ATP production, further supporting the hypothesis that mitochondrial remodeling in response to PI3K signaling is distinct from the mitochondrial effects of Akt signaling in the heart.
Our experiments in neonatal rat cardiomyocytes identify a potential Akt-independent target that may mediate the PI3K-dependent metabolic effects on cardiomyocytes. PKCλ/ζ was an attractive target from a metabolic standpoint as atypical PKC isoforms have been shown to play a role in GLUT4 translocation in muscle (Bandyopadhyay et al., 2000
; Etgen et al., 1999
) and have distinct effects from Akt on lipid metabolism in the liver (Matsumoto et al., 2003
; Taniguchi et al., 2006
). In our neonatal rat cardiomyocyte experiments we found a strong correlation between citrate synthase (CS) activity and cell number. These results indicate that in neonatal myocytes, growth factor signaling promotes a proliferative response that occurs in concert with a coordinate increase in mitochondrial content, whereas in the intact heart a coordinated increase in mitochondrial function accompanies myocyte hypertrophy. We defined conditions in which growth factor stimulation coordinately increased CS activity and cellular hyperplasia despite inhibition of Akt activation. By contrast, inhibition of growth-factor activation of PKCλ/ζ signaling led to a significant decrease in mitochondrial CS activity in the absence of any change in cellular viability. At the concentrations used, these inhibitors were relatively specific for their respective substrates although we cannot completely rule out off target effects. Nevertheless, these observations raise the possibility that PKCλ/ζ signaling may be one potential PIP3
/PDK1 downstream signal that is required for PI3K dependent mitochondrial remodeling, and provides preliminary evidence that alternative PI3K targets can influence mitochondrial function in response to growth factor stimulation. These data do not rule out possible roles for other PIP3
targets, such as serum- and glucocorticoid-regulated kinase (SGK), p90 ribosomal S6 kinase (RSK), p21-activated kinase 1 (PAK1), Rac which can modulate actin remodeling (Han et al., 1998
) or PLCγ which activates pathways downstream of IP3
and DAG (Xie et al., 2005
). PI3K significantly affects a broad range of cellular signaling pathways in cardiac muscle, and more work will be needed to establish all of the Akt-independent but PIP3-dependent signaling molecules that are required for regulation of mitochondrial remodeling in response to PI3K activation.
In summary, the current study identifies a central role for PI3K in coordinating physiological cardiac growth and metabolic capacity in the heart. Inhibition of PI3K is sufficient to prevent the mitochondrial adaptations to exercise-induced hypertrophy, despite increases in PGC-1α mRNA and protein levels, indicating that up-regulation of PGC-1α expression and PI3K activation may work in parallel to increase myocardial mitochondrial oxidative capacity during physiological cardiac growth. Additionally, whereas previous studies have identified an obligate requirement of PI3K activation of Akt1 as a key regulator of physiological cardiac cellular growth (DeBosch et al., 2006
; McMullen et al., 2003
), here we show that PI3K likely activates Akt-independent signaling pathways to mediate the metabolic and mitochondrial adaptation that accompanies physiological cardiac hypertrophy.