The liver transiently accumulates TAG following a single exercise bout, particularly if the exercise bout is challenging. This ability to rapidly expand the hepatic TAG pool size, referred to here as lipogenic flexibility, has been discovered previously but remains poorly understood. The present report describes observations of lipogenic flexibility, based upon findings from lipidomics analysis of the liver following a moderate-intensity exercise bout (CE) and a high-intensity exercise bout (HIIE) in mice. Through the present work, the presence of transient liver TAG accumulation after exercise has been rigorously confirmed, and the findings have been elaborated through the analysis of various lipid classes in the liver. Associations between liver TAG content and other lipids in CON mice were discovered, and these associations were transiently altered by a single exercise bout; the implications of these findings are discussed below. Discussion below also addresses intensity-dependence of the lipogenic flexibility response, as indicated by robust responses particularly to HIIE for TAG content changes as well as additional metabolic impacts indicated by PCA plots of acyl chain compositions. Finally, the overall implications of the present results are discussed in relation to the mammalian exercise response and the implications for metabolic health.
The exercise-responsive lipogenic flexibility in the liver is depicted primarily by a significant accumulation of hepatic TAG after exercise, which returns back toward baseline CON levels by the following day. Accurate measurement of TAG is truly essential for work aimed at characterizing this phenomenon. The investigative team authoring this report have observed this hepatic TAG phenomenon by two independent analytical procedures, including a colorimetric biochemical TAG assay published previously (17) and an analytical chemistry-based approach using thin layer chromatography followed by gas chromatography as reported in the present report (Table 1). Both approaches led to the observation of an intensity-dependent accumulation of TAG in the liver of female mice after exercise which resolved by the following day. In the biochemical work published previously, both sexes were studied, while for the current lipidomics approach a single sex was selected for sample submission to a lipidomics analysis laboratory. In these female mouse liver samples, there was a high degree of correlation between TAG results from the two methods, with a slope near unity; thus, one can be confident that previous observations are robust and confirmed by the present TAG analysis. What’s more, in the present report a broad range of lipids are reported, leading to an elaboration of the lipogenic flexibility phenomenon beyond the measurement of TAG and its lipid droplet-related proteins that were reported previously (17).
The present results in Figure 1A indicate that under sedentary conditions, there is a significant positive correlation between hepatic TAG and DAG content. This finding is consistent with previous knowledge that TAG accumulation in the liver is associated with DAG accumulation (1-5), which can then lead to impairments in metabolic health (2, 7, 8). However, when transient TAG accumulation is triggered in the liver by exercise, the correlation between TAG and DAG is disrupted (Figure 1B); this indicates that the metabolically inert TAG pool can accumulate in this scenario without being strongly associated with lipotoxic DAG accumulation. As the hepatic TAG content returned back toward CON levels during recovery on the day after exercise, its association with DAG content was also nearly reestablished. Transient enhancement of perilipin-2 (pln2) expression in the liver after exercise may stabilize lipid droplets as the TAG pool size expands (17). The previous results for pln2 expression (17) indicate enhanced capacity after exercise to transiently sequester TAG within hepatic lipid droplets, while DAG residing outside lipid droplets would not be sequestered by pln2.
Under sedentary conditions (CON), hepatic TAG is also significantly correlated with hepatic PE (Figure 2A), while this statistical association is disrupted during the rapid expansion of the hepatic TAG pool following a single exercise bout (Figure 2B). This is followed by a trend toward the correlation between TAG and PE being reestablished in exercised mice on the day after the exercise bout (Figure 2C). The positive association between TAG and PE in sedentary mice (CON) or exercise-recovered mice could be potentially expected, because cellular PE content could affect lipid droplet biology. PE is a component of the phospholipid monolayer surrounding lipid droplets (28) and may be involved in formation (29) or fusion of lipid droplets (30). Thus, a relationship between TAG accumulation and tissue PE content seems understandable. However, during the transient TAG accumulation in the liver that occurs rapidly in response to exercise, the TAG pool expansion outpaces any PE biosynthesis.
When considering the variety of lipid classes analyzed in this study, it is clear that TAG was the most responsive to exercise. This indicates that the lipidomic response to exercise in the liver may be primarily related to fuel metabolism rather than changes in structural lipids within the cell. While TAG was the only lipid class showing a change in concentration, DAG was the lipid class that showed a change in the distribution of fatty acid classes within the esterified lipid pool, with exercise-induced increases in total PUFA and n-6 PUFA alongside the corresponding reduction in SFA content. While these statistically significant changes in DAG composition may be reasonably modest in magnitude, it is noteworthy that they occurred in response to only a single bout of exercise, and they were sustained even the day following exercise. The mice in the study reported here consumed the 5K52 diet which contains a reasonably substantial n-6 PUFA abundance (46% of fatty acids) (25). After HIIE the PUFA content in liver DAG rose to approach this value of PUFA expected from the diet, which may be caused by an exercise-induced release of dietary fatty acids that were stored in adipose tissue. Furthermore, while discussing the impacts of exercise on hepatic lipids, it should be noted that the most substantial impacts were following HIIE, which is an exercise approach that exhibits particularly potent impacts upon health and metabolism (17, 26, 31-36). The remodeling of PUFA content in DAG occurred after HIIE but not CE, and the response of TAG concentration was enhanced following HIIE in comparison to CE. Furthermore, principal component analysis indicated that the separation between HIIE and CON to be more notable than separation between CE and CON, suggesting potentially a more potent impact upon turnover of cellular lipids in the liver. While PCA plots, even with an individual mouse as an outlier, indicated a general separation between HIIE and CON for the lipid classes reported (TAG, DAG, PE), the CE data points were broadly dispersed and overlapped with the CON group. As a whole, the data are supportive of a biologically distinct impact of HIIE in comparison to CE, even when these exercise types are matched for distance, duration, and energy expenditure. Thus, it appears that intermittently challenging exercise is more metabolically potent in the liver than sustained mild exertion.
In order to understand the metabolic events that lead to exercise-induced changes in hepatic lipids, the timing of the changes in TAG accumulation could be considered. In this work liver tissue was collected 3 h after exercise and the following day. The accumulation of hepatic TAG seen at 3 h after exercise hypothetically could have occurred during exercise, during the first few hours of post-exercise recovery, or during both time periods. There have been some reports indicating that TAG has already accumulated in the liver at the end of the exercise bout in humans (11, 13, 15), mice (37), and rats (24). In another study on human subjects, TAG accumulation did not occur during exercise but subsequently accumulated during four h of recovery (15); a similar observation was made studying mice, in which hepatic TAG did not accumulate during exercise but subsequent accumulated during three h of recovery (38). In contrast, in a study on rats hepatic TAG accumulated during exercise but began to recover soon after, substantially returning toward baseline even within an hour of recover (9). Alternatively, in mice the hepatic TAG that accumulated during exercise was fully maintained 3 h after exercise (37). Overall, it appears that TAG can potentially accumulate in the liver both during exercise and during hours following exercise, with a sustained elevation typically lasting for hours, but with recovery time ranging from 1 h to perhaps approximately 24 h. Nutritional status likely plays a role, and investigation of the effects of food/beverage intake after exercise deserves attention in the future. If accumulation of TAG in the liver is driven by plasma FFA concentration, then accumulation could be promoted both during and after exercise; exercise indeed leads to increased plasma FFA turnover and concentration both during exercise and during hours of post-exercise recovery (20). As discussed in the Introduction, control of lipolysis during and after exercise may have evolved based upon the fuel supply needs of skeletal muscle. However, as enhanced lipolysis drives an elevated FFA concentration in plasma and thus increased FFA uptake down concentration gradients into working muscle (39-41), this response places a metabolic burden upon non-contracting muscle and other organs such as the liver that will be presented with circulating FFA levels that are beyond their needs. It appears that enhanced circulating FFA, though useful for fuel trafficking from storage sites to sites of use, can place a burden and enhanced lipotoxicity risk upon peripheral tissues. Ideally, for preservation of metabolic health, this elevated FFA would be buffered into the TAG pool intracellularly, which is metabolically inert, rather than being stored in lipotoxic intermediate pools such as DAG.
While it appears plausible that lipogenic flexibility in the liver after exercise is driven largely by uptake of plasma FFA down concentration gradients, it is worth noting that the liver also possesses an elaborate network of enzymatic control of lipid metabolism. For example, diet can exhibit potent effects upon the lipogenic pathway through sterol regulatory element-binding protein 1c SREPB-1c (42, 43). This pathway leads to expression changes of lipogenic enzymes such as fatty acid synthase, stearoyl CoA desaturase, and acetyl CoA carboxylate (42, 43). Also diet can chronically modulate expression and activity of peroxisome proliferator activated receptor-α (PPAR-α) which impacts expression of enzymes related to the fat oxidation pathway (42-44) and mitochondrial function (44, 45). Chronic exercise training can improve mitochondrial function in the liver through effects on sirtuin expression, regulation of enzyme activities and substrate selection, or through mitochondrial biogenesis (46). Chronic exercise training may limit steatosis through actions upon these lipogenic and oxidative pathways, but it is expected that remodeling of mitochondrial metabolism would require many exercise bouts over a significant time period. The acute effect of each exercise bout would likely act through other means that are distinct from these impacts of diet and chronic exercise. Despite presence of a control network acting chronically through modulation of transcription factors, substrate supply also remains an important controller of tissue TAG synthesis and accumulation. When plasma FFA concentration is elevated, as occurs during and after exercise, hepatic TAG accumulation is expected because of enhanced FFA uptake. Furthermore, the enhanced pln2 expression in the liver after exercise (17) may impair access of lipolytic enzymes to the hepatic lipid droplets, promoting transient TAG accumulation. To summarize, exercise may very rapidly alter intracellular TAG content through pathways that do not necessarily require changes in lipogenic or fat oxidation enzyme expression levels.
It is understood that the liver after exercise is able to exhibit a lipogenic flexibility, supported by pln2 expression, that allows rapid expansion of the TAG pool and buffering of FFA into this inert pool (17). Next, it would be useful to consider this phenomenon exhibited by the liver in the context of lipid changes occurring in other tissues in response to exercise and related stressors. During exercise, the amount of intramuscular TAG declines in the exercised muscles, while TAG tends to accumulate elsewhere. TAG accumulates in the liver (present results) and even in skeletal muscle that was not actively recruited for the exercise bout (12). During fasting, which also stimulates lipolysis but is not associated with vigorous muscle contraction, skeletal muscle actually accumulates TAG as seen in laboratory animals by biochemical analysis (47) and in human subjects by measuring intramyocellular lipid by MRS (48-52). As with exercise, fasting leads to accumulation of TAG in the liver as observed in rodent studies (53-56) and accumulation of intrahepatocellular lipid (presumably mostly TAG) as observed by non-invasive MRS in human subjects research (57). It is important to keep in mind that the acute response to each bout of a stressor is not necessarily qualitatively similar to the chronic stress response. Specifically, chronic caloric restriction typically reduces hepatic TAG concentration (58) while acute fasting leads to elevation of hepatic TAG (57). Similarly, in some cases chronic exercise training modestly reduces hepatic TAG (6, 19); however, each acute bout of exercise transiently raises hepatic TAG (11, 13-16), even when pre-exercise hepatic TAG is high as in patients with non-alcoholic fatty liver disease (NAFLD) (14). Specifically, the exercise modalities reported here acutely raise hepatic TAG on the day of exercise, but in mice that were chronically trained by CE or HIIE, with liver tissue collection on the day following the last exercise bout, hepatic TAG content in exercised mice was not elevated above CON (26). While chronic adaptations are certainly meaningful, the ability to buffer excess plasma FFA into the hepatic TAG pool is likely to be metabolically critical when a stressor is acutely applied that increases circulating FFA.
STUDY STRENGTHS AND LIMITATIONS
The present study was designed to elucidate effects of acute exercise bouts upon subsequent resting metabolism. This investigation into the post-exercise recovery period benefited from inclusion of two time points. Three h after exercise depicts the early recovery phase (Day 1) while the time point on the follow day (Day 2) represents the late recovery phase as metabolism returned back toward baseline. It is a strength that a time-matched CON group was included, as it is critical to control for diurnal variation and changes in energy balance across the day. It is also a strength of the study that two different exercise types were tested, such that the findings could have implications for multiple exercise prescription options. It may be advantageous in future investigations to include earlier time points as well (such as collecting tissues immediately after exercise) in order to add additional temporal resolution to the findings. Secondly, while in this work non-obese female mice were studied, it would be useful to study both sexes as well as obesity when further exploring the topic of lipogenic flexibility in the future.