Exercise endurance capacity is markedly reduced due to impaired energy homeostasis during prolonged fasting in FABP4/5 deficient mice
Skeletal muscle prefers carbohydrate use to fatty acid (FA) use as exercise intensity increases. In contrast, skeletal muscle minimizes glucose use and relies more on FA during fasting. In mice deficient for FABP4 and FABP5 (double knockout (DKO) mice), FA utilization by red skeletal muscle and the heart is markedly reduced by the impairment of trans-endothelial FA transport, with an increase in glucose use to compensate for reduced FA uptake even during fasting. We attempted to determine whether prolonged fasting affects exercise performance in DKO mice, where constant glucose utilization occurs.
A single bout of treadmill exercise was performed in the fed and fasted states. The initial speed was 10 m/min, and gradually increased by 5 m/min every 5 min up to 30 m/min until the mice stopped running. Running distance was significantly reduced by DKO genotype and prior fasting, leading to the shortest distance in fasted DKO mice. Levels of glycogen in skeletal muscle and the liver were nearly depleted in both WT and DKO mice during prolonged fasting prior to exercise. Levels of TG in skeletal muscle were not reduced by exercise in fasted DKO mice, suggesting that intramuscular TG was not utilized during exercise. Hypoglycaemia was accelerated in fasted DKO mice, and this acceleration could be due to constant glucose utilization by red skeletal muscle and the heart where FA uptake is diminished due to defective trans-endothelial FA transport. Taken together, energy supply from serum and storage in skeletal muscle were very low in fasted DKO mice, which could lead to a significant reduction in exercise performance.
FABP4/5 have crucial roles in nutrient homeostasis during prolonged fasting for maintaining exercise endurance capacity.
KeywordsMetabolism Exercise Fasting Fatty acid Glucose Skeletal muscle Liver FABP4 FABP5
Carnitine palmitoyltransferase 1b/2
Fatty acid binding protein
Glucose-6-phosphate catalytic subunit
Glyceraldehyde 3-phosphate dehydrogenase
Glucose transporter 1/4
3-hydroxy-3-methylglutaryl-CoA synthase 2
Long-chain acyl-CoA dehydrogenase
Medium-chain acyl-CoA dehydrogenase
Myosin heavy chain
Non-esterified fatty acid
Peroxisome proliferator activated receptor γ coactivator 1 alpha
Peroxisome proliferator activated receptor α
Peroxisome proliferator activated receptor δ
Fatty acid binding proteins (FABPs) are 14–15 kDa cytosolic proteins that can reversibly bind to hydrophobic molecules such as saturated and unsaturated fatty acids (FAs) and eicosanoids with high affinity [1, 2]. It has been reported that FABPs promote the transport of lipids to specific compartments in the cells. Among FABPs, FABP4 (also referred to as A-FABP/aP2/ALBP) and FABP5 (also known as E-FABP/mal1) are abundantly expressed in adipocytes, macrophages and muscle-type capillary endothelial cells [1, 2, 3, 4, 5]. A series of studies with mice lacking both FABP4 and FABP5 (double knockout (DKO) mice) provided evidence that FABP4/5 are involved in the development of metabolic diseases, including diet-induced obesity, type 2 diabetes and insulin resistance [6, 7, 8]. Although it has been reported that both local and systemic inflammatory processes mediated by adipocyte and macrophage FABP4/5 can cause such metabolic diseases [1, 2, 6, 7, 9, 10], it remains obscure how the simultaneous disruption of FABP4/5 in mice exhibits remarkable effects on the amelioration of metabolic diseases.
Recent studies from our laboratory and others have reported that capillary endothelial FABP4/5 have an important role in FA transport via circulation into the heart and skeletal muscle (referred to as trans-endothelial FA transport) [3, 11, 12, 13]. A tracing study using 125I-BMIPP (FA tracer) and 18F-FDG (glucose tracer) revealed that DKO mice exhibit reduced FA uptake with a marked increase in glucose use in red/oxidative skeletal muscle and the heart even during fasting. Thus, trans-endothelial FA transport occurs in highly oxidative tissues with muscle type continuous capillaries, while a remarkable increase in glucose use is induced in the same tissues by a compensatory mechanism independently of insulin. These findings suggest that the ameliorating effects of FABP4/5 disruption observed in diseased models could be due to a compensatory increase in glucose use by the peripheral tissues, at least in part.
In contrast to the beneficial effects on metabolic diseases, DKO mice also exhibit an impairment of systemic metabolism when fasting is prolonged. In the fasted state in general, most tissues, except red blood cells and the brain, rely on FA use to produce energy and minimize glucose consumption [14, 15]. When fasting is prolonged, the hydrolysis of triacylglycerol (TG) in adipose tissue is accelerated, leading to an increase in plasma levels of non-esterified FA (NEFA) as the major energy substrate [14, 15]. Glycogen content in the liver becomes nearly depleted after a 24-h fast, while gluconeogenesis is accelerated to supply glucose to glucose-dependent peripheral tissues and cells [14, 15]. On the other hand, in DKO mice in the fasted state, plasma levels of NEFA are more elevated due to reduced FA uptake, which results in hepatosteatosis and hyperketonaemia . In addition, the compensatory use of glucose in red skeletal muscle and the heart leads to severe hypoglycaemia, which is also at least partly caused by a reduction in gluconeogenesis . Moreover, thermogenesis severely declines in fasted DKO mice when they are exposed to cold environment . Major energy substrates for thermogenesis, such as glycogen in skeletal muscle, TG in brown adipose tissue and plasma glucose, become less available because they are depleted rapidly in DKO mice, which results in fatal hypothermia . Thus, in DKO mice, metabolic adaptation to prolonged fasting is severely impaired due to defective FA utilization by red skeletal muscle and the heart, a compensatory use of glucose and the deranged redistribution of energy storage.
Exercise intensity influences the preference for energy substrates by skeletal muscle. When exercise intensity is low, the primary fuels are NEFA, released from adipose tissue via lipolysis, and glucose, derived from oral ingestion or the liver (gluconeogenesis or glycogenolysis) [18, 19]. As exercise intensity increases, the use of plasma glucose progressively increases, whereas the utilization of circulating NEFA declines. Simultaneously, the contribution of muscle glycogen progressively increases as well. When moderate intensity exercise continues, the contribution of the lipid oxidation in skeletal muscle is enhanced [18, 19]. Thus, exercise at a high-intensity enhances the relative contribution of carbohydrates, whereas exercise at a low to moderate intensity promotes the combustion of both carbohydrates and lipids as fuels [18, 19].
Metabolic homeostasis is impaired in DKO mice in response to fasting as described above. In this study, therefore, we studied whether exercise endurance is impaired more severely in DKO mice in the fasted state because energy provision to skeletal muscle is likely to be impaired during fasting. We found that in addition to reduced FA uptake, blood glucose levels and intramuscular energy storage were markedly decreased during fasting, which could cause a significant reduction in exercise endurance capacity in DKO mice. Our study suggests that FABP4/5 have crucial roles in nutrient homeostasis during prolonged fasting for maintaining exercise endurance capacity.
Animals and sample preparation
FABP4 and FABP5 double knockout (DKO) mice with the C57BL6j background were generated as described previously . Control male wild-type (WT) mice with the C57BL6j background were purchased from Japan SLC Inc. one week before treadmill exercise training. The age (10 to 12 weeks) and body weight (22 to 27 g) of WT and DKO mice were comparable. The mice were housed and fed as described previously . To reduce pain for euthanasia, the mice were briefly anaesthetized in an isoflurane-filled box to induce early unconsciousness, and then maintained using a mask type isoflurane inhalation system during blood sampling. Blood was collected from the retro-orbital plexus, and then centrifuged at 1500×g for 15 min at 4 °C to obtain the serum for measurement of biochemical parameters. After cervical dislocation, samples of the liver and skeletal muscle were dissected, snap frozen in liquid nitrogen and stored at − 80 °C until further use. All study protocols were approved by the Institutional Animal Care and Use Committee (Gunma University Graduate School of Medicine). Animal experiments were performed according to the NIH guidelines (Guide for the Care and Use of Laboratory Animals).
Treadmill exercise testing
To determine the exercise endurance capacity, treadmill exercise testing was carried out using a 2-lane motorized rodent treadmill (MK-680, Muromachi Kikai, Tokyo, Japan) [20, 21]. Mice were accustomed to the treadmill running twice, and then a single bout of running was carried out as described previously (Fig. 2b) [20, 21]. When mice stopped running more than 3 s on the electric shocker, we defined the time point as exhaustion. The total running distance for each mouse was calculated.
Measurements of glycogen and triacylglycerol
Liver and skeletal muscle were snap frozen in liquid nitrogen and then powder-pulverized. Glycogen and TG were extracted and then measured as described previously using the glycogen assay kit (BioVision, CA) and the Triglyceride E-test Wako (Wako Chemical, Osaka), respectively [3, 16].
Quantitative analysis of mRNA expression
Total RNA was isolated from liver and skeletal muscle and quantitative real time-PCR were carried out as described previously [3, 17]. The expression levels of the target genes were normalized to the level of glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA. The Perfect Real Time Primers (ready-to-use gene-specific primers purchased from Takara Bio Inc., Shiga, Japan) were used.
Determination of myosin heavy chain isoforms in skeletal muscle
Isoform expression of myosin heavy chain (MHC) in the soleus was determined by an immunofluorescence as described previously . Primary antibodies against MHC I (BA-F8) and MHC IIa (SC-71) were purchased from the Developmental Studies Hybridoma Bank (University of Iowa). Secondary antibodies (Alexa Fluor 350 IgG2b for MHC I and Alexa Fluor 555 IgG1 for MHC IIa) were purchased from Invitrogen. Wheat germ agglutinin conjugated with Alexa Fluor 488 (Invitrogen) was used to stain plasma membrane of each muscle fibre. A combination of type IIb and IIx fibres was recognized as muscle fibres negative for MHC I and MHC IIa. Muscle fibres positive or negative for each MHC isoform were counted and presented as percentage of fibre types.
IBM SPSS (version 24 for Windows, IBM, NY, USA) was used for statistical analysis. The results are shown as the mean ± standard deviation. A two-way analysis of variance (ANOVA) was utilized to determine the effects of feeding status (fed or fasted) on running distance between WT and DKO mice (DKO genotype). A three-way ANOVA was utilized to analyse interaction effects on gene expression and metabolites levels among exercise, DKO genotype or feeding status. P < 0.05 was considered significant. *p < 0.05, **p < 0.01, ***p < 0.001.
A remarkable reduction in running distance in fasted FABP4/5 DKO mice
Accelerated hypoglycaemia in fasted DKO mice
Accelerated depletion of energy storage in fasted DKO mice
Expression of genes for gluconeogenesis and ketogenesis in liver was comparable between WT and DKO mice in the fasted state
We next studied the mRNA expression levels of genes related to β-oxidation and ketogenesis, which were regulated by PPARA [23, 24]. The expression of Ppara and β-oxidation related genes (Cpt1a/2, Lcad and Mcad) was induced by fasting and partially affected by exercise, but not influenced by DKO genotype (Fig. 4b), suggesting that β-oxidation activity was not affected in DKO mice when compared to WT mice. The expression of Hmgcs2, a rate-limiting enzyme in ketogenesis, was increased by fasting in both WT and DKO mice (Fig. 4c). Thus, there was no enhancement of mRNA expression for gluconeogenesis or ketogenesis in the livers of fasted DKO mice during exercise, which may cause early exhaustion due to no compensatory supply of alternative energy substrates from the liver to the skeletal muscle.
Expression of genes for energy combustion in skeletal muscle was not reduced in fasted DKO mice
Composition of fibre types in oxidative skeletal muscle was comparable
We demonstrated that FABP4/5 have a critical role in exercise endurance in the fasted state. Energy storage, such as glycogen in the liver and glycogen and TG in skeletal muscle, were almost depleted in fasted DKO mice. Accelerated utilization of glucose in oxidative muscle and the heart is maintained even during fasting and gluconeogenesis is diminished , which could result in accelerated hypoglycaemia. Further, FA uptake is decreased in oxidative muscle due to defective trans-endothelial FA transport via capillary endothelial FABP4/5 [3, 16]. As a result, energy supply from serum and energy storage in skeletal muscle are very low in fasted DKO mice prior to exercise, which could bring about a significant reduction in exercise performance. We conclude that deranged uptake and redistribution of energy substrates during prolonged fasting provoke maladaptive energy homeostasis, leading to a significant reduction in exercise endurance capacity.
It is noteworthy that exercise performance was not reduced much in DKO mice compared to WT mice in the fed state (Fig. 1c) when glycogen and TG storage was fulfilled (Fig. 3b and c). We recently reported similar findings in CD36 knockout (KO) mice . Like FABP4/5 DKO mice, CD36KO mice also display a reduction in FA transport and oxidation with a compensatory use of glucose [26, 27]. In CD36KO mice, endurance capacity is severely reduced in the fasted state, while the reduction is modest in the fed state . These findings further support the notion that intact FA utilization by peripheral tissues plays an important role in nutrient homeostasis in the fasted state to maintain the exercise endurance capacity. In contrast to our observation, however, previous studies showed a reduction in exercise endurance capacity in CD36KO mice even during fasting [28, 29, 30]. However, between our studies and previous reports with CD36KO mice, there is a major difference regarding the exercise protocols. The treadmill speed used in previous studies was 17 m/min or less (mild to moderate intensity) [28, 29, 30]. In our studies, the exercise speed was increased up to 30 m/min in a single bout (high intensity). High-intensity exercise induces maximum reliance on glycogen in skeletal muscle, while mild to moderate intensity exercise leads to modest reliance on intramuscular glycogen and TG, as well as circulating NEFA . In addition to energy reliance, mild to moderate intensity exercise results in a longer exercise time (hours), which could, in turn, augment the relative contribution of FABP4/5/CD36-mediated FA uptake from the circulation. Thus, exercise intensity, as well as duration, affects the preference for main energy substrates for endurance, which can cause differences in the endurance capacity between WT mice and mice with defective FA utilization. In the fed state, endurance capacity for high intensity exercise is more affected by glycogen content in skeletal muscle, whereas the capacity for mild to moderate intensity exercise is more influenced by the FA uptake ability.
In addition to an enhanced reduction in endurance capacity, we reported that DKO mice exhibit lethal hypothermia in response to cold exposure in the fasted state . Accelerated hypothermia during fasting is mainly caused by a marked reduction in TG storage in brown adipose tissue, glycogen in skeletal muscle and blood glucose. Importantly, rapid hypothermia does not occur in the fed state, when energy storage in brown adipose tissue and skeletal muscle is filled. Thus, an inappropriate redistribution of energy substrates occurs in DKO mice only in the fasted state, which leads to the depletion of tissue energy storage for thermogenesis and exercise. Similar responses during fasting were also observed in CD36KO mice, another model with defective FA utilization [20, 32], suggesting that such physiological disadvantages during fasting seem to be a common feature in animals with defective FA utilization. These findings further provide an important notion that in general, appropriate FA utilization by peripheral tissues is essential to adapt to severe natural environments such as famine and coldness, and to chase animals or escape from natural enemies during fasting.
There is accumulating evidence that the gut microbiota plays a role in the host metabolic status as a mediator of dietary intake [33, 34]. Germ-free mice have reduced adiposity, improved tolerance to glucose and insulin and are protected from diet-induced obesity when fed a Western diet [33, 34]. It has also been suggested that an altered composition of the microbiota is associated with increased host adiposity via a number of different mechanisms [33, 34]. In addition, the mouse microbiota is known to be heavily affected by their environment. In this study, control WT mice were purchased from an outside vendor one week before treadmill exercise training, while DKO mice were born and grown in our animal facility. Thus, the difference of housing conditions between WT and DKO mice might affect the results of metabolic data to a certain degree, which needs careful interpretation.
In our previous studies, we estimated cardiac function in DKO mice in pressure overload-induced and lipopolysaccharide-induced cardiomyopathy models [35, 36]. Cardiac dysfunction was more obvious in DKO mice compared to WT mice in both models, presumably due to energy depletion. Interestingly, however, induction levels of mRNA for natriuretic peptide b (Nppb), a marker of cardiac stress, were comparable in WT and DKO mice, suggesting that cardiac dysfunction occurs by energy depletion with no change of Nppb expression levels (unpublished observation). In this study, we also found comparable induction of Nppb in WT and DKO mice after exercise in the fasted state. Based on our previous observation, the result does not necessarily mean that cardiac function is similar between WT and DKO mice. Thus, the possibility that a reduction in exercise endurance capacity in DKO is caused by cardiac dysfunction remains unsolved.
FA uptake by peripheral tissues via capillary endothelial FABP4/5 has crucial roles in nutrient homeostasis for maintaining exercise endurance capacity during prolonged fasting.
We thank Takako Kobayashi and Keiko Matsukura for excellent technical help. We thank Drs. Hiroshi Yorifuji and Hitoshi Ueno for technical help with the treadmill exercise.
This work including collection and analysis of data was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 16H05294 to MK and no. 17 K09568 to TI), a grant from the Gunma University Initiative for Advanced Research (no. 140908272400 to MK), and a grant from the Japan Agency for Medical Research and Development (no. 10030–14120 to MK).
Availability of data and materials
The datasets used and/or analysed in the current study are available from the corresponding author upon reasonable request.
TI designed this study. HH, HS, MM, HM, YU, MRAAS, TY and TI collected physiological and biochemical data via treadmill exercise. HH and RT performed the immunofluorescence. HH and TI analysed all the data. TI and MK contributed to the manuscript writing. The final version of the manuscript was approved by all authors.
Ethics approval and consent to participate
All study protocols were approved by the Institutional Animal Care and Use Committee (Gunma University Graduate School of Medicine) and were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
Consent for publication
The authors declare that they have no competing interests.
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