To test this hypothesis, we first examined the effects of succinate on skeletal muscle fiber composition, metabolism, and exercise tolerance. By combining pharmacological and siRNA‐mediated knockdown model both in vitro and in vivo , we demonstrated that succinate induces skeletal muscle transition from fast twitch to slow twitch through the SUCNR1 signaling pathway. Our results indicate potential use of succinate as a dietary supplement to improve physical fitness and counteract fatigue.

Endurance or aerobic exercise is crucial to muscle fiber‐type remodeling by increasing the mechanical and metabolic demand on skeletal muscle 9 . Previous study showed endurance training increases intracellular calcium concentration ([Ca 2+ ] i ) 10 11 , which activates the calcineurin/nuclear factor of activated T cells (NFAT) 12 13 and myocyte enhancer factor‐2 (MEF2) 14 . These two transcription factors play a dominant role in muscle formation and fiber remodeling. In addition to transient elevation of [Ca 2+ ] i , endurance exercise also increases several specific TCA cycle intermediates, among which succinate increases the most 15 16 . However, whether these intermediates mediate endurance exercise‐induced muscle fiber transition is rarely investigated. Succinate has been reported to induce cardiomyocyte hypertrophy 17 and osteoclastogenesis 18 . It also plays an important role in energy 19 and glucose 20 homeostasis by regulating mitochondrial oxygen consumption 21 and heat production from brown adipose tissue (BAT) 22 . Therefore, we hypothesize that succinate regulates skeletal muscle fiber remodeling.

In mammals, skeletal muscle comprises about 55% of the individual body mass 1 2 . Skeletal muscle is heterogeneous and composed of slow‐ and fast‐twitch fiber types, which differ in contractile‐protein composition, oxidative capacity, and substrate preference for ATP production 3 . Slow‐twitch fibers have more myoglobin, more mitochondria 4 , a higher level of intracellular calcium concentrations 5 , and higher activity of oxidative metabolic enzymes than fast‐twitch fibers. Therefore, the switching of skeletal muscle fiber from fast twitch to slow twitch is important for sustained and tonic contractile events 6 7 , maintenance of energy homeostasis 8 , and alleviation of fatigue.

Male C57BL/6J mice were injected with LV‐shScrambled or shSUCNR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After 2 weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. (A) Cumulative food intake, (B) body weight gain, (C) fat mass, and (D) lean mass of mice after 6 weeks of dietary SUC supplementation.

Male C57BL/6J mice were injected with LV‐shScrambled or shSUCNR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After 2 weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks.

Since SUCNR1 is universally expressed in most metabolic tissues, including adipose tissue, liver, and heart, succinate may indirectly act on SUCNR1 expressed in other metabolic tissues to regulate skeletal muscle metabolism and fiber switching. To exclude this possibility, we further generated and validated a gastrocnemius‐specific SUCNR1 knockdown mouse model by gastrocnemius‐specific injection of SUCNR1 siRNA lentivirus during adulthood (Fig 8 A and B). Consistent with our observation in congenital SUCNR1 global knockout mice, SUCNR1 selective knockdown in the gastrocnemius muscle showed no effects on food intake (Fig EV5 A), body weight (Fig EV5 B), and body composition (Fig EV5 C and D). Importantly, SUCNR1 gastrocnemius‐specific knockdown consistently attenuated the regulatory effect of succinate on exercise capacity (Fig 8 C–E), muscle fiber type (Fig 8 I and J), and related associated enzyme activity (Fig 8 F–H). Together, these data support an indispensable role of muscle SUCNR1 in succinate‐induced skeletal muscle fiber remodeling.

Schematic representation of SUCNR1 KO by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) strategy. The sgRNA sites were located in intron 1 and intron 2 of SUCNR1 gene. Four sgRNAs were designed to delete exon 2 of SUCNR1 gene. The DNA sequences contained sgRNA‐binding regions are labeled with lines.

To determine the role of SUCNR1 in succinate‐induced skeletal muscle fiber switching in vivo , we constructed a congenital SUCNR1 global knockout mouse model (Fig EV4 A–C). We found that SUCNR1 null mice showed the same body weight gain (Fig EV4 G), food intake (Fig EV4 D), and body composition (Fig EV4 E and F) as their wild‐type littermates. Additionally, we found the stimulatory effects of succinate on AKT/mTOR/FOXo3a pathway were diminished in SUCNR1 KO mice (Fig EV4 H and I), suggesting a SUCNR1‐mediated activation on protein synthesis. Interestingly, SUCNR1 KO also effectively blocked the regulatory effects of succinate on oxygen consumption (Fig 7 A and B), RER (Fig 7 C and D), and exercise capacities, including slow‐speed running time (Fig 7 G), four‐limb handing time (Fig 7 F), and muscle grip (Fig 7 E). Consistently, the activities of SDH, HK, and LDH (Fig 7 H–J); skeletal muscle fiber type (Fig 7 K–N); and the expression of NFAT and PGC‐1α (Fig 7 K and L) failed to be changed by succinate in the gastrocnemius of SUCNR1 KO mice. These data support an essential role of SUCNR1 in succinate‐induced skeletal muscle fiber switching.

After 6 days of differentiation, C2C12 cells were treated with vehicle, SUC (2 mM), U73122 (5 μM), or SUC (2 mM) + U73122 (5 μM) for 48 hrs. Representative images (C) and (D, E) quantification of MyHC I and MyHC IIb immunofluorescent staining (green) in the C2C12 cells ( n = 3). Scale bar in (C) represents 50 μm.

To test this point of view, we generated pharmacological or genetic loss‐of‐function models to investigate the requirement of SUCNR1 in succinate‐induced muscle fiber‐type transition. We found that succinate triggered a transient elevation of [Ca 2+ ]i in C2C12 myotubes (Fig 6 D) and promoted nucleic NFAT accumulation in the gastrocnemius muscle shortly (0.5–3 h) after acute succinate administration (Fig 6 E–G). Importantly, pharmacological blockage of PLC‐β, a key mediator of a GPCR‐triggered calcium signaling pathway, effectively abolished succinate‐induced [Ca 2+ ]i elevation (Fig EV3 A) and fiber‐type transition (Fig EV3 B–D) in C2C12 myotubes. Consistently, siRNA‐mediated knockdown of SUCNR1 in C2C12 myotubes (Fig 6 H) effectively abolished succinate‐induced [Ca 2+ ]i elevation (Fig 6 I); myotube fiber conversion (Fig 6 M–O); activity changes in SDH, HK, and LDH (Fig 6 J–L); and lactate production (Fig EV3 E). These in vitro data suggest that succinate‐induced C2C12 myotube fiber switch is mediated by SUCNR1.

To explore the intracellular mechanism for succinate‐induced fiber‐type transition, we compared the expression of SUCNR1, an endogenous receptor of succinate 25 , in the soleus and gastrocnemius muscles. Interestingly, the protein (Fig 6 A and B) and mRNA (Fig 6 C) of SUCNR1 in the soleus (typical slow/slow muscle) are much higher than levels in gastrocnemius (typical mixed slow/fast muscle). In addition, exercise significantly increased SUCNR1 protein expression in both soleus and gastrocnemius muscles (Fig 6 A and B), suggesting a potential role of this receptor in skeletal muscle fiber‐type remodeling.

Additionally, we tested the number, morphology, and activity of mitochondria. Consistent with our in vivo data, succinate significantly increased mitochondrial DNA content (Fig 5 F), cellular mitochondrial density (Fig 5 G–J), and coverage (Fig 5 I). However, the size (Fig 5 J) and the membrane potential of mitochondria (Fig EV2 B and C) were not affected by succinate. These results suggest that the enhanced aerobic oxidation is mainly due to the increased mitochondrial number, but not the activity of each mitochondrion. These in vitro data reveal a direct role of succinate in the slow‐twitch transition, mitochondrial biogenesis, and aerobic oxidation.

To test the direct effect of succinate on skeletal muscle, we used C2C12 myotubes as an in vitro model to study the role of succinate in skeletal muscle fiber‐type remodeling. Similar to the previous in vivo study, we found that succinate significantly increased the proteins and genes of slow‐twitch fiber markers, while decreased the proteins and genes of fast‐twitch markers as indicated by both immunofluorescence (Fig 5 A and B) and qPCR (Fig EV2 A). Regarding metabolic enzymes, succinate enhanced the activity of SDH (Fig 5 C), but reduced the activity of LDH (Fig 5 D) and lactic acid production (Fig 5 E) in C2C12 myotubes.

Based on a recent study showing that succinate increased adipose tissue metabolism and induced browning in high‐fat diet (HFD)‐induced obesity mice 22 , we postulated that succinate has a similar stimulatory effect on metabolism in muscle. To test this, we further evaluated oxygen consumption in skeletal muscle and consistently found that succinate significantly increased oxygen consumption ratio (OCR) in the gastrocnemius (Fig 4 L). Together, these results indicate that succinate induces skeletal muscle fiber remodeling by promoting mitochondrial biosynthesis and aerobic oxidation.

Male C57BL/6J mice were fed with chow diet supplemented with 0 and 1% SUC for 6 weeks.Data information: Results are presented as mean ± SEM (= 4–6). Different letters between bars mean0.05 in one‐way ANOVA analyses followed byTukey's tests. *0.05 and **0.01 by non‐paired Student's‐test.

Immunoblots and quantification of p‐AMPK, PGC‐1α, and myoglobin in gastrocnemius. The same lysates were used for the detection of PGC1α (100 kDa, Fig 4 I), myoglobin (17 kDa, Fig 4 I), myosin heavy chain (180 kDa, Fig 3 B), and tubulin (48 kDa, shared in both Figs 3 B and 4 I).

A high number of mitochondrial and metabolic adaptation are generally accompanied with endurance exercise and skeletal muscle type transition 24 . Here, we tested the effects of succinate on metabolism and mitochondrial properties. We found that succinate increased whole‐body oxygen consumption (Fig 4 A and B) and decreased whole‐body respiratory exchange ratio (RER; Fig 4 C and D) in the dark cycle. In addition, serum non‐essential fatty acid (NEFA) content was decreased by succinate supplementation (Fig 4 E), suggesting that the decreased respiratory quotient may be attributed to the elevated fatty acid oxidation. Consistently, succinate enhanced the activity of succinate dehydrogenase (SDH; Fig 4 F) and hexokinase (HK; Fig 4 G) but suppressed the activity of lactic dehydrogenase (LDH; Fig 4 H). These results suggest that succinate promotes aerobic metabolism. In supporting this point of view, an enhanced mitochondrial biogenesis was consistently shown in our model. When detecting the myosin heavy chain by WB, we also checked PGC1α and myoglobin protein simultaneously (Fig 4 I and J), as well as the expression of genes related to mitochondria and electron transport chain (Fig 4 K). These protein and mRNA expression level were dose‐dependently increased by succinate in the gastrocnemius. However, the p‐AMPK levels were reduced by succinate (Fig 4 I and J), indicating that cellular energy status may not be the main reason for skeletal muscle type transition.

Oxidative capacity of three muscles was also evaluated by the staining of succinate dehydrogenase (SDH), a marker of oxidative capacity of skeletal muscle at the fiber level. We found that succinate dose‐dependently increased the percentage of SDH‐positive fibers in SOL, EDL, and gastrocnemius muscles (Fig EV1 I–N), suggesting succinate is sufficient to improve mitochondrial content and oxidative capacity of mixed (gastrocnemius), slow/slow (SOL), or fast/fast (EDL) muscles.

Consistently, we found that succinate dose‐dependently increased MyHC I but not MyHC IIb protein expression in soleus, suggesting an increased proportion of slow‐twitch fiber (Fig EV1 E and F). On the other hand, succinate failed to affect the muscle fiber composition of EDL muscle (Fig EV1 G and H).

In mixed gastrocnemius muscle, we found that succinate upregulated slow‐twitch fiber‐associated genes MyHC I, MyHC IIa, PGC‐1α, myoglobin, and TnnT1, whereas it downregulated fast‐twitch fiber‐associated genes, including MyHC IIb and TnnT3 (Fig 3 A). Further, both Western blot (Fig 3 B) and immunofluorescence (Fig 3 C and D) demonstrated that succinate increased MyHC I/IIa protein expression and slow‐twitch fiber percentage, while decreased MyHC IIb protein and fast‐twitch fiber percentage. These results indicate that succinate induces a fast twitch to slow‐twitch transition in skeletal muscle.

There are four types of skeletal muscle fiber, including I, IIa, IIx, and IIb. Each of them expresses different myosin heavy chain and troponin isoforms. Here, we studied the effects of succinate on muscle fiber‐type transaction in three different muscles, including soleus, extensor digitorum longus (EDL), and gastrocnemius. Soleus is known as a typical slow‐twitch muscle (slow/slow), whereas EDL is a typical fast‐twitch muscle (fast/fast). Gastrocnemius usually has a lot of fast‐twitch muscle fibers, or an equal number of fast and slow‐twitch fibers (fast/slow mixed).

It is well‐known that endurance exercise performance is determined by oxygen supply and muscle fiber type 19 . We first tested if the oxygen‐carrying capacity of muscle was enhanced by succinate. We found although succinate slightly increased the number of red blood cells (RBC; Fig 2 E) and the hemoglobin (HGB) level (Fig 2 F), the extent of these increases is not comparable to the dramatic improvement of endurance exercise capacity. In order to further characterize other parameters related to endurance exercise capability, we used an ex vivo strategy to evaluate isolated muscle contraction properties (Fig 2 G). We found that dietary supplementation of succinate did not affect the maximum contractile force (Fig 2 I), but significantly improved fatigue resistance of muscle (Fig 2 H and J), with less glucose consumption (Fig 2 K), and lactate production (Fig 2 L) during contraction. Taken together, our data indicate that succinate can increase oxygen‐carrying capacity and reduce muscle fatigue.

To further investigate the effects of succinate on skeletal muscle contraction properties, we first tested the exercise capacity of mice. We found that succinate dose‐dependently increased muscle grip strength (Fig 2 A), low‐speed running time (Fig 2 B), and decreased falling time in four‐limb handing test (Fig 2 C). However, high‐speed running time was unchanged by succinate supplementation (Fig 2 D), which indicates succinate may specifically improve endurance exercise performance, but not explosive exercise performance.

The percentage of SDH positive in the (I, J) gastrocnemius, (K, L) soleus, and (M, N) extensor digitorum longus muscle is shown by SDH enzyme staining. Only darkly stained SDH fibers are treated as SDH‐positive fibers. The graphs show the SDH‐positive fiber ratios ( n = 4–6). Scale bar in I, K, and M represents 100 μm.

Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5, and 1% SUC for 8 weeks.Data information: Results are presented as mean ± SEM (= 6–8). Different letters between bars mean0.05 in one‐way ANOVA analyses followed byTukey's tests. *: significant difference (0.05) between 0.5% SUC and control group by non‐paired Student's‐test. #: significant difference (0.05) between 1% SUC and control group by non‐paired Student's‐test.

To determine the effects of succinate on skeletal muscle growth, we fed male C57BL/6J mice with chow diet supplemented with 0, 0.5%, or 1% succinic acid disodium salt for 8 weeks. We found that succinate‐supplemented diet increased serum SUA level (Fig 1 A) but had no effects on the body weight gain (Fig 1 B), food intake (Fig EV1 A), fat mass (Fig 1 C), lean mass (Fig 1 D), gastrocnemius muscle index (Fig 1 E), or liver index (Fig EV1 B). Additionally, consistent with our previous report 23 , we found that succinate activated Akt/mTOR cascade and inhibited FoxO3a (Fig EV1 C and D). Interestingly, we also found that 1% succinate increased the proportion of small muscle fiber (200–400 μm 2 ), while decreased the proportion of large muscle fiber (600–800 μm 2 ; Fig 1 F and G). This shift of muscle fiber size distribution indicates that succinate may affect skeletal muscle contraction properties.

Discussion

Skeletal muscle fiber types are distinguished by myosin heavy chain (MyHC) isoforms 26, metabolic enzyme activity 6, mitochondrial number 27, and contractile properties 28. Endurance or aerobic exercise is well known as an effective way to induce skeletal muscle remodeling by increasing mechanical and metabolic demand on skeletal muscle 293031. Interestingly, exercise also dramatically elevates the content of several TCA cycle intermediates, including succinate 32. Succinate previously has been shown to regulate mitochondrial function and reactive oxygen species production in muscle 33, which is a distinguishing feature of skeletal muscle fiber types 34. Based on these observations, we speculate that succinate is a key mediator for exercise‐induced muscle fiber remodeling.

In supporting this point of view, we found that dietary succinate supplementation improved the endurance exercise performance and attenuated skeletal muscle fatigability, accompanied by enhanced aerobic metabolism and upregulated MyHC I/IIa expression. These data demonstrated for the first time that succinate induces a switch from fast twitch to slow‐twitch fibers, suggesting a potential mechanism for metabolite‐mediated skeletal muscle fiber‐type transition.

Mitochondria are the main sites of cellular aerobic respiration. In general, cellular or tissue oxidative metabolism is enhanced by increasing the number of mitochondria 35. PGC‐1α has been shown to be a key regulator of mitochondrial biosynthesis and oxidative metabolic enzyme 36. Overexpression of PGC‐1α increases mitochondrial content and the oxidase levels of skeletal muscle, which results in more resistance to fatigue 37. In this study, we found that succinate increased the protein expression of PGC‐1α, as well as the mitochondrial content both in vitro and in vivo. In addition, succinate further enhanced O 2 uptake in skeletal muscle cells. This observation is consistent with a previous study showing that succinate increases mitochondrial oxygen consumption in ex vivo skeletal muscle obtained from septic animals.

Besides the number of mitochondria, the function of mitochondria was also strengthened by mitochondrial membrane potential and mitochondrial membrane enlargement 38. Thus, we further examined the morphology changes in mitochondria by succinate using an electron microscope. We found that succinate increased mitochondrial number without changing mitochondrial size. Although we were unable to examine the function of all signal mitochondria, enzyme activities and O 2 uptake strongly suggested that the increase in mitochondrial number is accounted for the enhanced mitochondrial function in skeletal muscles.

Skeletal muscle fiber‐type remodeling involves several key signaling pathways, including calcium 39 and AMPK 40. In this study, we found that succinate boosted [Ca2+] i and increased the protein expression of calcineurin, MEF2, and NFATc1 in skeletal muscles. MEF2 and NFATc1 are important transcription factors for skeletal muscle fiber switching 41. When translocated from the cytoplasm to the nucleus, NFAT regulated calcium‐dependent target genes that promoted the formation of slow muscle fibers 42. Another important muscle remodeling pathway is Ca2+/CaMK, which increases MEF2, thereby promotes the formation of slow‐twitch fiber types 4344. Ca2+ played a dominant role in these two signaling pathways 45. Thus, we wondered if Ca2+ mediated succinate‐induced fiber‐type switch in muscle.

To test this hypothesis, we blocked [Ca2+] i by inhibiting PLC‐β and found that succinate‐induced fiber‐type transition was effectively abolished by PLC‐β antagonist. These results demonstrated that succinate‐induced muscle fiber transition was closely associated with calcium signaling pathway and its downstream transcript factors, MEF2 and NFATc1. On the other hand, we found that succinate decreased p‐AMPK/AMPK ratio, suggesting an increased intracellular energy state. The decreased AMPK activity might be attributed to the enhanced oxidative capacity and ATP production. This evidence indicated that AMPK signaling pathway might not be involved in succinate‐induced skeletal muscle fiber‐type transition.

Besides acting as a metabolite in the TCA cycle, succinate also exhibits a hormone‐like function through the activation of G‐protein‐coupled receptor SUCNR1 46. SUCNR1 is expressed throughout the whole body 4748 and has been reported to couple with either Gi or Gq protein to trigger different intracellular pathways 49. For example, succinate elevates the levels of hemoglobin, platelets, and neutrophils 50 and enhances immunity 51 through SUCNR1‐coupled G i ; it also increases intracellular calcium 52 coupled with Gq to release arachidonic acid along with prostaglandins E2 and I2. Here, we showed that succinate increased the expression of SUCNR1 and its downstream factor PLCβ, which were associated with boosted [Ca2+] i . This finding suggests that succinate may act on Gq‐coupled SUCNR1 in skeletal muscles. In supporting this view, SUCNR1 global knockout or selective knockdown in skeletal muscle abolished the regulatory effects of succinate on muscle fiber transition both in vitro and in vivo. Our data demonstrated that SUCNR1 is the primary mediating receptor for the effect of succinate on skeletal muscle fiber‐type remodeling.

Consistent with our previous report on the stimulatory effects of succinate on protein synthesis in skeletal muscle 23, we also found dietary supplementation of succinate activated Akt/mTOR cascade and inhibited FoxO3a in WT mice. These regulatory effects of succinate were diminished in SUCNR1 KO mice, suggesting a SUCNR1‐mediated activation on protein synthesis. In this context, a seemingly paradoxical finding is that dietary supplementation of succinate failed to increase muscle mass. How can succinate increases skeletal muscle protein synthesis without changing muscle weight? We speculate that this inconsistency may be due to succinate‐induced muscle type remodeling from fast‐ to slow‐twitch fibers. It is known that slow‐twitch fibers have lower fiber size and higher oxidative proteins and capacity for protein synthesis compared to fast‐twitch fibers 53. Succinate‐induced hypertrophy of skeletal muscle may be neutralized by the discrepancy in fiber size of slow‐ and fast twitch or mass of large myofibrillar proteins and much smaller oxidative proteins. Alternatively, it is also possible that the protein synthesis is balanced by a high rate of protein degradation resulting in a higher turnover rate in the high oxidative fibers.

Regular exercise and chronic hypoxia are natural stimuli that produce sustainable cardioprotection against ischemia reperfusion 54. Consistent with the important role of succinate in muscle metabolism and fiber remodeling we showed, succinate is elevated in the blood in response to exercise 32 and accumulated rapidly in hypoxic/ischemic tissues 335556, suggesting a potential role of succinate in exercise/hypoxia‐mediated cardioprotection. Succinate may act as a paracrine or endocrine signaling molecules via SUCNR1 to regulate local cellular metabolism 57, or increase tissue blood supply through the renin‐angiotensin system, thereby alleviating tissue hypoxia and hypoxia adaptation of metabolism in the environment 585960. Consistently, augmentation of succinate has been shown to improve cardiac ischemic energetics, a source of damage at reperfusion 55. Therefore, succinic acid may not only play an important role in autocrine regulation of skeletal muscle metabolism and fiber‐type conversion, but also improve the adaptability of cardiovascular and brain tissues to the ischemic environment.

Our results demonstrated that dietary succinate supplementation led to remodeling of muscle fiber without changing body weight or fat distribution, suggesting that the primary function of succinate is to regulate muscle type transition but not body weight. However, our study was carried out under normal chow diet (low‐fat diet), which may have concealed a phenotype relevant for human obesity normally induced by high‐energy/fat diet. Indeed, a recent study has shown that water supplementation of 1.5% but not 1% succinate stimulates uncoupling protein 1 (UCP1)‐dependent thermogenesis from BAT, which induces robust protection against HFD‐induced obesity 22. This discrepancy suggests a diet‐dependent anti‐obesity effect of succinate, which may be attribute to different baseline UCP1 activation in chow and HFD condition. It has been shown that HFD significantly inhibits the expression and metabolic activity of UCP‐1 in BAT 61. The inconsistency may also be due to different supplementary method and dose (1.5% in water vs. 1% diet). The effective dose of succinate to remodel skeletal muscle fiber type may be lower than that to reduce body weight and fat mass.

In conclusion, our results demonstrated that succinate induces a SUCNR1‐mediated transformation from fast‐ to slow‐twitch fiber types in skeletal muscle. This finding indicates the potential application of succinate as exercise mimetics for people who are bedridden or disable to maintain their fitness, and even for athletes to improve their performance. Additional studies are warranted to identify the high‐affinity ligands of SUCNR1, which may be helpful to maintain muscle energy homeostasis and alleviate fatigue.