Bioenergetic function plays a role in various chronic diseases including cardiovascular disease, diabetes, and some neurodegenerative disorders. In these instances, bioenergetic function is thought to constitute a viable therapeutic target (Fontan-Lozano et al. 2008; Ren et al. 2010; Duncan 2011; Swerdlow 2011). Exercise, a non-pharmacologic intervention, affects cell and tissue bioenergetics. While exercise's effects on muscle bioenergetics are particularly robust these effects are not muscle-limited and, at least to a minor degree, occur in other tissues. It was recently postulated that physical exercise might potentially delay or mitigate age-related central nervous system diseases such as Alzheimer's disease (AD), perhaps through effects on brain bioenergetics (Rockwood and Middleton 2007).
In muscle, exercise facilitates mitochondrial biogenesis (Hood et al. 2006), and this confers some benefits of endurance training. Exercise may also affect brain mitochondrial biogenesis. One study found that exercise increases brain peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1α) mRNA levels (Steiner et al. 2011). In several tissues, PGC-1α acts as a master regulator of mitochondrial biogenesis and cell energy metabolism. It binds and activates nuclear respiratory factor 1 (NRF-1), which in turn induces the expression of mitochondrial transcription factor A (TFAM) (Scarpulla 2008; Vina et al. 2009). TFAM enables the replication, maintenance, and transcription of mitochondrial DNA (mtDNA). Under some conditions, exercise has been shown to increase brain mtDNA copy number (Bayod et al. 2011; Marosi et al. 2012; Zhang et al. 2012a,b).
Exercise has long been thought to primarily modify brain molecular physiology by increasing the amounts of brain-derived neurotrophic factor (Stranahan et al. 2009), but other factors may also mediate the non-muscle effects of exercise, or perhaps lie upstream of brain-derived neurotrophic factor changes. Lactate, which is generated and released by exercising muscle, in particular appears to affect the brain. Blood lactate accesses the brain via endothelial monocarboxylate transporters (MCTs) (Pierre and Pellerin 2005). Lactate imported from the blood to the brain is used to generate energy (Quistorff et al. 2008; Gallagher et al. 2009; van Hall et al. 2009; Boumezbeur et al. 2010; Wyss et al. 2011), protects ischemic neurons (Berthet et al. 2009), and facilitates memory formation (Newman et al. 2011; Suzuki et al. 2011). For these reasons and others, we considered whether lactate itself might reproduce and perhaps mediate exercise-associated changes in brain bioenergetic infrastructures.
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- Materials and methods
Our study shows supra-lactate threshold exercise and lactate itself in some ways similarly alter brain and liver bioenergetic infrastructures. Overlapping changes involve the expression of genes that monitor, respond to, and modify cell bioenergetic states. Our data suggest that in the liver, exercise-generated lactate enhances gluconeogenic infrastructure while reducing expression levels of some proteins that maintain respiration-associated infrastructures. In the brain, exercise-generated lactate enhances PRC and VEGF expression and does not down-regulate respiration-related infrastructures. In general, in the liver and brain, lactate itself to some extent functions as an exercise mimetic. These findings have clinical and clinical trial-design implications.
In this study, hepatic gluconeogenesis data come from mice exercised above the lactate threshold. Our current findings are consistent with a previous study in which mice exercised below the lactate threshold (E et al. 2013). The liver plays a central role in the Cori Cycle, in which exercise-generated lactate is removed from the blood and used to generate glucose. It is not surprising, then, that the expression of two gluconeogenic genes, PCK1 and PDK4, increased in the livers of EX and LAC mice and that mRNA levels of PGC-1α, which promotes hepatic gluconeogenic gene expression (Herzig et al. 2001), also increased.
PGC-1α is the most studied PGC-1 family member; the effects of exercise on PGC-1β and PRC are not well known. In general, PGC-1β drives gluconeogenic gene expression less robustly than PGC-1α, but is a stronger activator of NRF-1 and inducer of respiration-relevant gene expression (Lin et al. 2002, 2003). PGC-1β, therefore, likely supports cell respiration to a greater extent than PGC-1α.
The liver, of course, helps to avoid hypoglycemia. To counter hypoglycemia under conditions of increased glucose utilization, a state that exercise may induce, the liver imports lactate from the blood, converts it to glucose, and releases glucose into the blood. An increase in hepatic respiration could theoretically compete with this role. Avoiding this competition could perhaps explain why exercise associates with increased liver PGC-1α expression, but decreased PGC-1β expression.
We also explored the ability of exercise and lactate to modify the expression of bioenergetics-associated genes in the brain. Lactate certainly appears to play a critical role in brain energy metabolism (van Hall et al. 2009; Boumezbeur et al. 2010; Wyss et al. 2011), and while most brain lactate is regionally generated by astrocytes in response to local metabolic activity, externally generated lactate accesses the brain via MCTs (Pierre and Pellerin 2005). In this study, we found supra-lactate threshold exercise and intraperitoneal lactate injections both increase brain VEGF-A mRNA expression. This suggests exercise-generated lactate could at least partly mediate exercise's ability to increase brain VEGF-A mRNA levels. VEGF-A expression is also elevated within tumor cells, which frequently overproduce lactate (Siemeister et al. 1998; Walenta and Mueller-Klieser 2004). The possibility that tumor-generated lactate might contribute to increased tumor VEGF-A expression seems worth considering.
VEGF is believed to play a critical role in exercise-induced neurogenesis (Fabel et al. 2003; Tang et al. 2010; Latimer et al. 2011). In general, this signal protein binds specific cell membrane tyrosine kinase receptors (VEGFRs), and this binding stimulates angiogenesis (Cross et al. 2003). Exercise induces VEGF expression in mouse and rat brains, where it drives dentate gyrus, cerebellum, and hippocampus angiogenesis (Black et al. 1990; Fabel et al. 2003; Pereira et al. 2007). Angiogenesis in general is activated by hypoxia, which initiates HIF-1α nuclear translocation, and HIF-1 complex formation activates VEGF transcription (Pugh and Ratcliffe 2003). While one study found intense exercise-reduced brain mitochondrial O2 (Secher et al. 2008), because HIF-1α mRNA and protein levels did not change we cannot implicate hypoxia as the cause of our observed brain VEGF increase.
PGC-1α, interestingly, increases VEGF expression via estrogen-related receptor alpha co-activation (Arany et al. 2008). While PGC-1α expression was not increased in either EX or LAC mouse brains, we did not assess PGC-1α protein levels or activation states. We therefore cannot conclude PGC-1α did not mediate VEGF expression.
mRNA levels of PRC, another PGC-1 family member, increased in both EX and LAC mouse brains. PRC also interacts with estrogen-related receptor alpha to induce mitochondria-related gene transcription, and in general supports mitochondrial ATP production (Vercauteren et al. 2009; Mirebeau-Prunier et al. 2010). Although it is not currently known whether PRC specifically stimulates VEGF expression, we observed a positive correlation between mouse brain PRC and VEGF-A mRNA expression. This correlation suggests PRC may play a role in mediating exercise and lactate-associated VEGF expression changes.
Unlike hepatocytes, which use lactate to support gluconeogenesis, neurons mostly use lactate to support respiration (Pellerin and Magistretti 1994; Erlichman et al. 2008; Gallagher et al. 2009; Wyss et al. 2011). While down-regulating respiration infrastructure in the liver may have physiologic advantages, it is difficult to envision situations in which down-regulating respiration infrastructure would benefit the brain. Perhaps for this reason, brain PGC-1β, NRF-1, and TFAM expression did not decline with either supra-lactate threshold exercise or the administration of exogenous lactate.
Our EX mice failed to increase brain PGC-1α mRNA expression, although Steiner et al. previously reported exercise can increase brain PGC-1α expression (Steiner et al. 2011). In that study, mice exercised on a treadmill 1 h per day, at 25 m/min and a 5% incline, for 8 weeks. Differences between our exercise protocol and that of Steiner et al. may account for this inter-study discrepancy. Our lactate injection data, though, suggest exercise-generated lactate did not drive the brain PGC-1α mRNA increase observed by Steiner et al.
Similarly, we could not prove that lactate mediates an exercise-associated increase in brain mtDNA. While mtDNA content was higher in EX mice than it was in SED mice, mtDNA levels between LAC and VEH mice were equivalent. Although our study joins several others that also found exercise increases brain mtDNA copy number (Bayod et al. 2011; Steiner et al. 2011; Marosi et al. 2012; Zhang et al. 2012a,b), what causes this phenomenon remains unclear. In addition to arguing that lactate is not solely responsible, our data further suggest PGC-1α expression changes are not a prerequisite.
As we found in a study of mice exercised below the lactate accumulation threshold (E et al. 2013), mice exercised above the lactate accumulation threshold have lower brain TNF-α expression. LAC mice did not show reduced brain TNF-α mRNA, which suggests lactate does not account for this change. The exercise-related mechanism that drives reduced brain TNF-α expression remains unknown.
In this study, exercise training profoundly enhanced lactate thresholds and exceeding this threshold was not, from an exercise protocol perspective, straightforward. This suggests that over the long term, exercising human subjects with chronic illnesses above the lactate threshold will be impractical. If it turns out that intensive exercise benefits the brain more than mild or moderate exercise, exercise mimetics will likely be needed.
It is worth considering whether lactate itself could serve as a clinically useful exercise mimetic. Over the 2-week lactate treatment period mild weight loss occurred; it remains unclear whether this reflects an adverse or exercise mimetic-like effect. Lactate, it is reported, may stimulate beta cell insulin secretion, suppress glycolysis, and cause insulin resistance (Meats et al. 1989; Choi et al. 2002). Although lactate treatment in our study increased blood glucose measurements, plasma insulin levels decreased and HOMA-IR values certainly did not rise.
In contrast to our lactate treatment HOMA-IR data, even though exercise is known to reduce insulin resistance our EX mice had elevated HOMA-IR values. This was driven by increased fasting insulin levels. Other investigators report that in mice exercise training increases islet insulin secretion (Huang et al. 2011), although the underlying basis of this phenomenon remains unclear. Also, the HOMA-IR value primarily provides an estimate of liver insulin resistance (Bonora et al. 2000). For this reason and others, we do not believe exercise truly increased overall insulin resistance in our EX mouse group.
For our two experimental groups, the exercised mice and the lactate-injected mice, we did not attempt to exactly match the pharmacokinetic parameters of brain and liver lactate exposure. Maximum levels and daily exposure durations were almost certainly different between these groups, and the number of days of increased lactate exposure definitely differed. This was partly a consequence of feasibility limitations, as accomplishing this would have required us to repeatedly sample mouse plasma lactate levels while the mice were running on the treadmill. Our goal, though, was simply to test whether administering lactate itself induces some of the same bioenergetic infrastructure and bioenergetic-related changes that exercise induces. Our data argue it does.
We did not specifically test whether lactate affects liver and brain bioenergetic pathway flux dynamics in different or similar ways, although our molecular-based data predict differences should outweigh similarities. Unlike the liver, the brain is not considered a gluconeogenic organ. Neurons do not express glucose-6-phosphatase, the enzyme that mediates the final step of gluconeogenesis (Bell et al. 1993; Lehninger et al. 2005). Although astrocytes do express glucose-6-phosphatase, astrocytes dephosphorylate glucose at much lower rates than they phosphorylate glucose. This contrasts with liver-derived observations (Bhattacharya and Datta 1993; Gotoh et al. 2000; Ghosh et al. 2005). Further, astrocytes express the MCT4 monocarboxylate transporter isoform, which favors lactate export over import, whereas neurons express MCT2 transporters that favor lactate import (Pierre and Pellerin 2005). Overall, bioenergetic cross-talk between neurons and astrocytes seems designed to favor lactate production by astrocytes and lactate consumption by neurons. For these reasons, we suspect that while liver lactate exposure increases liver gluconeogenesis, increasing brain lactate levels should cause no or little increase in brain gluconeogenesis. This prediction, though, remains experimentally unconfirmed.
In conclusion, our results are consistent with the view that during exercise, muscle-generated lactate accounts for at least some exercise-associated brain and liver bioenergetic infrastructure and bioenergetic-associated adaptations. These adaptations are tissue-specific, are defined by each tissue's bioenergetic-related needs and obligations, and based on these needs and obligations are ultimately predictable. Lactate enhances hepatic gluconeogenesis while probably limiting hepatocyte respiration. Although lactate probably does not account for all major exercise-related, bioenergetics-associated brain changes, it does appear that it is able to induce brain VEGF expression and through this may drive brain angiogenesis and neurogenesis. For this reason, exercise mimetics that reproduce lactate's brain effects are worth developing for, and testing in, conditions with perturbed brain energy metabolism.