Figure 1.
Expression of PKC isoforms in HFD and exercised mice.
A, Expressions of PKC isoforms in liver. Wild-type (WT) mice fed on a high-fat diet (HFD) feeding were either exercised (EX) or sedentary (SED) for 8 weeks. Liver was isolated and used for Western blot detection of PKC isoforms (Top panel, representative images; bottom panel, statistical analysis). *, P<0.05. B, Expressions of PKC isoforms in skeletal muscle. WT mice fed on a HFD were either exercised or sedentary for 8 weeks. Skeletal muscle tissue was isolated and used for Western blot detection of PKC isoforms (Top panel, representative images; bottom panel, statistical analysis). *, P<0.05.
Figure 2.
Changes in body weight, food and water intake following HFD and exercise.
A, Effect of exercise on body weight of WT and PKCβ-/- mice. Mice were weighted once a week during the exercise-training period. B, Weight gain of WT and PKCβ-/- mice during the exercise-training period. Weight gain was less in WT mice but not in PKCβ-/- mice after exercise. C & D, Food intake (C) and water intake (D) of WT and PKCβ-/- mice with or without exercise. WT, wild-type; EX, exercise; SED, sedentary; Data are shown as mean ± SEM; n = 8 for each group; *, P<0.05 WT vs. PKCβ-/-; #, P<0.05 EX vs. SED.
Figure 3.
Changes in fat and muscle tissue after exercise in WT and PKCβ-/- mice.
A, Tissue weights in WT and PKCβ-/- mice with or without exercise. Muscle (thigh and calf) weights were increased and inguinal fat weight decreased in WT mice after exercise but not in PKCβ-/- mice. PKCβ-/- mice had higher muscle weight and lower fat weight compared to WT mice. B, Percent tissue weight over body weight in WT and PKCβ-/- mice with or without exercise. Exercise increased muscle percentage and decreased percentage of inguinal fat weight in WT mice but not in PKCβ-/- mice. PKCβ-/- mice had higher muscle weight and lower fat weight compared to WT mice with the same intervention. C, Representative images of magnetic resonance imaging (MRI) of abdominal cavity. D, Quantitative analysis of fat volume as determined by MRI. PKCβ-/- mice had significantly lower fat volumes of total, subcutaneous (SubQ), and visceral fats than WT mice and exercise did not significantly change it compared to sedentary (SED) intervention. WT, wild-type; EX, exercise; SED, sedentary; Data are shown as mean ± SEM; N = 8, *, P<0.05.
Figure 4.
Effect of exercise and PKCβ deficiency on insulin resistance.
A & B, Intraperitoneal glucose tolerance test (IPGTT) in WT and PKCβ-/- mice with or without exercise. A, Blood glucose responses; B, Area under the curve (AUC) of IPGTT. C & D, Intraperitoneal insulin tolerance test (IPITT) in WT and PKCβ-/- mice with or without exercise. C, Blood glucose curve; D, Area under the curve (AUC) of IPITT. E, Homeostasis model assessment-estimated insulin resistance (HOMA-IR) in WT and PKCβ-/- mice with or without exercise. HOMA-IR was calculated using the formula HOMA-IR = fasting glucose (mg/dl) x fasting insulin (µU/mL)/405. F, Fasting serum insulin level. After 16 h of fasting, serum was collected for ELISA detection of insulin. Exercise decreased fasting insulin level in WT but not in PKCβ-/- mice. Insulin level in sedentary PKCβ-/- mice was lower than that of sedentary WT mice. G, Fasting serum leptin level. After 16 h of fasting, serum was collected for ELISA detection of leptin. Exercise decreased fasting leptin level in WT but not in PKCβ-/- mice. Leptin level in sedentary PKCβ-/- mice was lower than that of sedentary WT mice. H, Fasting serum adiponectin level. After 16 h of fasting, serum was collected for ELISA detection of leptin. No significant difference of adiponectin was detected among the 4 groups. WT, wild-type; EX, exercise; SED, sedentary; Data are expressed as mean ± SEM; n = 8, #, P<0.05, WT vs. PKCβ-/-; $, P<0.05, EX vs. SED; *, P<0.05.
Figure 5.
Metabolism increased after exercise in WT but not in PKCβ-/- mice.
A, Resting O2 consumption over 24 hours. O2 consumption of WT and PKCβ-/- mice with or without exercise was measured in a resting state for 24 hours using a computer-controlled, Comprehensive Lab Animal Monitoring System (CLAMS). B, Average resting O2 consumption in WT and PKCβ-/- mice with or without exercise. C, Resting CO2 production over 24 hours. CO2 production of WT and PKCβ-/- mice with or without exercise was measured in a resting state for 24 hours at 22°C in presence of food and water using CLAMS. D, Average resting CO2 production in WT and PKCβ-/- mice with or without exercise. E, O2 consumption with exercise intervention. O2 consumption of WT and PKCβ-/- mice with or without exercise was measured. F, Average exercise O2 consumption in WT and PKCβ-/- mice with or without exercise. G, CO2 production with exercise intervention. CO2 production of WT and PKCβ-/- mice with or without exercise was measured. H, Average exercise CO2 production in WT and PKCβ-/- mice with or without exercise. WT, wild-type; EX, exercise; SED, sedentary; Data are expressed as mean ± SEM; n = 5, *, P<0.05.
Figure 6.
Both exercise and PKCβ deficiency reduced high-fat diet-induced mitochondrial dysfunction in the skeletal muscle.
A, Representative images of transmission electronic microscopy (TEM) show mitochondrial abnormality in the skeletal muscle of WT SED mice and reduced mitochondrial damage in the other 3 groups. Note the swollen and decreased matrix density of mitochondrial in WT SED group. Bar: 500 nm; Magnification: 18500×; The arrows indicate mitochondria. B, Mitochondrial number analysis shows that exercise increased mitochondrial number in WT but not in PKCβ-/- mice. Six images per mice, 3 mice per group have been counted and quantified. C, Mitochondrial size analysis shows that mitochondrial size was significantly increased in WT SED group but not in the other 3 groups. Six images per mice, 3 mice per group have been counted and quantified. WT, wild-type; EX, exercise; SED, sedentary; Data are presented as mean ± SEM; *, P<0.05.
Figure 7.
Effect of exercise and PKCβ deficiency on adipose tissue inflammation and plasma cytokines.
A–D, Adipose inflammation in WT and PKCβ-/- mice with or without exercise. Exercise slightly reduced the infiltration of macrophages although not statistically significant. Macrophage percentage in SVF was significantly lower in PKCβ-/- mice. Macrophages with M1 or M2 phenotype were not affected by exercise or PKCβ deficiency. A, Representative flow cytometric plots of adipose tissue macrophages; B, flow statistical analyses of adipose tissue macrophages; C, Percentage of classically activated macrophages (M1, CD11b+ CD11c+) in adipose tissue macrophages; D, Percentage of alternatively activated macrophages (M2, CD11b+ CD204+) in adipose tissue macrophages. E–G, Plasma cytokine levels in WT and PKCβ-/- mice with or without exercise. Plasma isolated from exercise or sedentary mice was collected for inflammatory cytokine detection using BD™ Cytometric Bead Array Mouse Inflammation Kit. Exercise and PKCβ deficiency do not affect the plasma level of IL-6, IL-10, and MCP-1. E, Plasma IL-6 level; F, Plasma IL-10 Level; G, Plasma MCP-1 Level. WT, wild-type; EX, exercise; SED, sedentary; Data are presented as mean ± SEM; n = 5, *, P<0.05.
Figure 8.
Effect of exercise and PKCβ deficiency on insulin signaling.
A, Insulin signal pathway in liver. Insulin downstream signal molecule AKT was activated after exercise in WT mice and had a less effect in PKCβ-/- mice (Top panel, representative images; bottom panel, statistical analysis). *, P<0.05. B, Insulin signal pathway in skeletal muscle. Insulin downstream signal molecule AKT was activated after exercise in WT mice and had a less effect in PKCβ-/- mice (Top panel, representative images; bottom panel, statistical analysis). *, P<0.05. WT, wild-type; EX, exercise; SED, sedentary; Data are presented as mean ± SEM; n = 3, representative bands from one of 3 independent experiments.