Fsp27 plays a crucial role in muscle performance

Abstract

Fsp27 was previously identified as a lipid droplet-associated protein in adipocytes. Various studies have shown that it plays a role in the regulation of lipid homeostasis in adipose tissue and liver. However, its function in muscle, which also accumulate and metabolize fat, remains completely unknown. Our present study identifies a novel role of Fsp27 in muscle performance. Here, we demonstrate that Fsp27^-/- and Fsp27^+/- mice, both males and females, had severely impaired muscle endurance and exercise capacity compared with wild-type controls. Liver and muscle glycogen stores were similar among all groups fed or fasted, and before or after exercise. Reduced muscle performance in Fsp27^-/- and Fsp27^+/- mice was associated with severely decreased fat content in the muscle. Furthermore, results in heterozygous Fsp27^+/- mice indicate that Fsp27 haploinsufficiency undermines muscle performance in both males and females. In summary, our physiological findings reveal that Fsp27 plays a critical role in muscular fat storage, muscle endurance, and muscle strength.NEW & NOTEWORTHY This is the first study identifying Fsp27 as a novel protein associated with muscle metabolism. The Fsp27-knockout model shows that Fsp27 plays a role in muscular-fat storage, muscle endurance, and muscle strength, which ultimately impacts limb movement. In addition, our study suggests a potential metabolic paradox in which FSP27-knockout mice presumed to be metabolically healthy based on glucose utilization and oxidative metabolism are unhealthy in terms of exercise capacity and muscular performance.

Keywords: Cidea; Cidec; diabetes; fat metabolism; lipid droplets; lipids; obesity.

Graphical abstract

Figure 1.

Weight, food, and water consumption of mice. Body weight (A; in g) and body-fat percentage (B) of wild type, Fsp27^+/− (Het-KO) and Fsp27^−/− (Fsp27-KO) mice. Food consumption over 96 h (C) for each mouse genotype in male mice. The right panel shows the quantification of total food consumption in 4 days. Tissue mass was weighed immediately after dissection in male (D) and female (E) mice. Water consumption over 96 h (F) for each mouse genotype. The right panel shows quantification of total water consumption in 4 days (n = 6 mice for each group). Data are expressed as means ± SE. For statistical analysis, one-way ANOVA followed by Tukey’s multiple comparison test was performed, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2.

Glucose and insulin tolerance testing in mice. Data are graphed over time for male and female mice. A: glucose insulin tolerance test (GTT) of males. B: the area under the curve (AUC) of GTT for males. C: insulin tolerance test (ITT) of males. D: AUC of ITT for males. E: GTT of females. F: AUC of GTT for females. G: ITT of females. H: AUC of ITT for females. I: fasted basal glucose, taken after overnight fasting, for both male and female mice. WT, wild type; Het-KO, Fsp27^+/− mouse; Fsp27-KO, Fsp27^−/− mouse (n = 6 mice for each group). Data are expressed as means ± SE. For statistics on A, C, E, and G, two-way ANOVA followed by Bonferroni post hoc analysis was performed; for statistics on B, D, F, and H, one-way ANOVA followed by Tukey’s multiple comparison test was performed, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

Metabolic phenotyping of each mouse genotype. Mice were monitored through metabolic chambering for a total of 5 days. Measurements were taken during the day cycle (6:00 AM to 6:00 PM) and the night cycle (6:00 PM to 6:00 AM) were averaged within each respective time cycle. The respiratory exchange ratio for day and night cycles (A), V̇o₂ (B), and V̇co₂ (C). Heat generation through indirect calorimetry (D). Het-KO, Fsp27^+/− mouse; Fsp27-KO, Fsp27^−/− mouse (n ≥ 6 male mice/group). Data are expressed as means ± SE. For statistics, one-way ANOVA followed by Tukey’s multiple comparison tests was performed, *P < 0.05, ***P < 0.001, ****P < 0.0001.

Figure 4.

Locomotor activity of each mouse genotype. Mice were monitored through metabolic chambering for a total of 5 days. Measurements were taken during the day cycle (6:00 AM to 6:00 PM) and the night cycle (6:00 PM to 6:00 AM) were averaged within each respective time cycle. Mouse movement along the X-axis, either ambulatory (A and B). Total X-axis movement (C and D). Z-axis movement (E and F). Het-KO, Fsp27^+/− mouse; Fsp27-KO, Fsp27^−/− mouse (n ≥ 6 male mice/group). Data are expressed as means ± SE. n ≥ 6 male mice per group. For statistics, one-way ANOVA followed by Tukey’s multiple comparison tests was performed separately for day and night cycle, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Muscle physiology testing and fiber typing. Treadmill running endurance was measured with an Exer 3/6 Metabolic Treadmill (A). After acclimation, mice (n >10 mice/group) were allowed to run until exhaustion as defined by spending over 5 consecutive s in the fatigue zone (see materials and methods). Results from the four-limb hanging grid test performed on mice (n >10 mice/group) with a minimum of 5 trials (B). Total hang time was used to calculate holding impulse after converting the weight of the mouse into Newtons. Fiber typing of the gastrocnemius (C), tibialis anterior (D), and extensor digitorum longus (E) muscle sections with multiplex immunofluorescent antibody staining (n = 3 male mice/group). WT, wild type; Het-KO, Fsp27^+/− mouse; Fsp27-KO, Fsp27^−/− mouse. Data are expressed as means ± SE. For statistics, one-way ANOVA followed by Tukey’s multiple comparison tests was performed separately for each fiber types, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

Lipid and glycogen analysis. Serum collected from fasted wild type and Fsp27^−/− mice (n = 5) was analyzed for total free fatty acids (FFAs) and triglycerides (TGs; A) as well as the lipid species composition (B–E). For A one-way ANOVA followed by Bonferroni post hoc analysis was performed. Data for the amount of each lipid species in the male TG fraction (B), lipid species in female TG fraction (C), lipid species in male FFA fraction (D), and lipid species in female FFA fraction (E). For B–E two-way ANOVA followed by Bonferroni post hoc analysis was performed. Liver (F) and muscle (G) glycogen content in fasted and unfasted wild-type and Fsp27^−/− male mice (n = 3 mice per group). WT, wild type; Fsp27-KO, Fsp27^−/− mouse. All data are expressed as means ± SE. For statistics, F and G one-way ANOVA followed by Tukey’s multiple comparison test was performed, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7.

Energy reserves, fuel utilization, muscle mass, and mitochondrial/hepatic output during exercise in WT and FSP27^−/− mice. Soleus muscle mass (A), gastrocnemius muscle mass (B). q-PCR analysis of mitochondrial genes normalized to β-actin (C). Western blot analysis of mitochondrial proteins: cytochrome C, COX IV, and voltage dependent anion selective channel 1 (D). Serum glucose levels (E), FFA levels (F), liver (G), and muscle (H) glycogen levels. Blood lactate levels before and after exercise in WT and FSP27^−/− animals (I). WT, wild type; FSP27^−/− male mice (n = 5 male mice/group). Data are expressed as means ± SE. For statistics, unpaired t test was performed for A and B, two-way ANOVA followed by Tukey’s multiple comparison test was performed for E–I. For C two-way ANOVA followed by Šídák’s multiple comparisons test was used to analyze the significance between the groups *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 8.

Localization of fat and Fsp27 in muscle. A: confocal microscopy images representing the localization of Fsp27 (magenta; Cy5), neutral lipid (green; BODIPY), and nuclei (blue; DAPI) in the gastrocnemius of wild type, Fsp27^+/− (Het-KO), and Fsp27^−/− (FSP27-KO) mice. B: qualitative measurement of fat staining in the gastrocnemius of wild type, Fsp27^+/−, and Fsp27^−/− mice. C: intramuscular triglyceride contents. n = 5 male mice/group. Data are expressed as means ± SE. For statistics one-way ANOVA followed by Tukey’s multiple comparison test was performed **P < 0.01 ***P < 0.001, ****P < 0.0001.