Figure 1.
Blood glucose is decreased while serum levels of FFA and ketone bodies are markedly increased during prolonged fasting in DKO mice.
Blood was collected from the vena cava inferior before (0-hr) and after fasting (24- and 48-hr) to measure blood glucose, NEFA, insulin, ketone bodies (BHB), triacylglycerol (TG) and cholesterol. n = 7−11/group. Blood was sampled from the retro-orbital plexus for plasma glucagon. n = 6/group. Data are shown as the mean ± SE. WT vs. DKO. ⋆p<0.05, ⋆⋆p<0.01, ⋆⋆⋆p<0.001.
Figure 2.
Locomotor activity is comparable between WT and DKO mice.
Locomotor activity was measured during feeding and fasting (0–24 and 24–48 hr). Data are presented for 12 hours of the light phase, 12 hours the dark phase and combined light and dark phase (24 hr).
Figure 3.TG.
accumulation is enhanced in DKO livers during prolonged fasting.
(A to E) Livers were isolated before (0 hour) and after fasting (24- and 48-hr). (A) Representative gross appearance of the liver. (B) Oil red O staining. Scale bar = 100 µm. (C) Triglyceride content in the liver (mg/mg protein). (D) Body weight (BW), % reduction in BW after fasting, liver weight (LW), ratio of LW relative to BW (LW/BW, mg/g). n = 11/group. (E) The expression of genes involved in FA uptake was determined using quantitative real-time PCR. (F and G) Mice received intravenous injections of 125I-BMIPP (5 kBq) and 18F-FDG (100 kBq) via the lateral tail vein before (0-hr) and after fasting (24- and 48-hr). The animals were sacrificed at 2 hours after injection. Uptake of 125I-BMIPP (F) and 18F-FDG (G) by the liver was quantified using a well-type gamma counter (n = 4). (H) Hepatocytes were isolated from WT and DKO livers. Uptake of 14C-palmitic acid (14C-PA) by hepatocytes was measured using a liquid scintillation counter. Note that uptake of 14C-PA was comparable between WT and FABP4/5 DKO hepatocytes. In addition, the uptake was proportional to the loading dose of 14C-PA. n = 6/group. Data are shown as the mean ± SE. WT vs. DKO. ⋆p<0.05, ⋆⋆p<0.01, ⋆⋆⋆p<0.001, ns = not significant.
Figure 4.
VLDL secretion is reduced in DKO mice after fasting.
(A) Serum levels of TG were measured at the indicated time points after an intravenous injection of triton WR 1339 (500 mg/kg). (B) The VLDL production rate (mg/kg/hour) was calculated from the TG concentration of (A). n = 5−8/group. (C) The expression of ApoB100 was determined using quantitative real-time PCR. n = 4/group. Data are shown as the mean ± SE.WT vs. DKO. ⋆p<0.05. (D) The expression of genes involved in FA synthesis was determined using quantitative real-time PCR.
Figure 5.
Rate of FA oxidation is enhanced in DKO mice.
(A and B) Livers were isolated before (0 hour) and after fasting (24- and 48-hr). (A) The expression of genes involved in FA metabolism was determined using quantitative real-time PCR. (B) The indicated metabolites from the livers of WT and DKO mice after 48 hr fast were measured using metabolome analysis. n = 7/group. Note that energy charge ((ATP+1/2ADP)/(ATP+ADP+AMP) = 0.51, more than 0.5 guarantees sampling quality) served as a quality control of material handlings [32], [33]. The lactate/pyruvate ratio was higher compared to previous reports [34], which might be due to technological differences between the enzymatic/chemical approach used in previous studies and the CE-MS approach used in this study. (C) Estimation of FAO using primary cultures of hepatocytes. n = 10/group. (D) Estimation of FAO using liver homogenates before (0-hr) and after fasting (24- and 48-hr). n = 6/group. Data are shown as the mean ± SE. WT vs. DKO. ⋆p<0.05, ⋆⋆p<0.01, ⋆⋆⋆p<0.001.
Table 1.
Metabolome analyses of DKO livers.
Figure 6.
Fasting-induced hepatic steatosis is reversible.
(A) DKO mice fasted for 48 hours. After resuming food intake, blood and liver samples were collected at the indicated time points (24-, 48- or 72-hr after refeeding). Mice that did not undergo fasting (0-hr) or experienced 48 hours of fasting were used as controls. The TG content in the liver and the serum levels of biochemical parameters (NEFA, ketone bodies, TG and glucose) were measured as previously described in the Materials and Methods section. n = 5−11/group. Data are shown as the mean ± SE. Control vs. no fasting/refeeding ⋆⋆p<0.01, ⋆⋆⋆p<0.001.
Figure 7.
Impaired glucose homeostasis in DKO mice during prolonged fasting.
(A to E) Livers were isolated before (0-hr) and after fasting (24- and 48-hr). (A) Glycogen storage in the liver of WT and DKO. n = 8/group (B) Representative PAS staining of the liver of the WT and DKO mice. Scale bar = 100 µm. (C and D) The indicated metabolites from the livers of WT and DKO mice after 48-hr fast were measured using metabolome analysis. n = 7/group. (E) The expression of genes involved in gluconeogenesis was determined using quantitative real-time PCR. (F) Estimated gluconeogenesis using the pyruvate challenge test. After 24 hour of fasting, pyruvate (2 gram/kg) was intraperitoneally injected. Blood was taken from the tail vein to measure the blood glucose levels at the indicated time points. n = 6/group. Data are shown as the mean ± SE. WT vs. DKO. ⋆p<0.05, ⋆⋆p<0.01, ⋆⋆⋆p<0.001.
Figure 8.
Ketogenesis is enhanced in DKO mice.
(A) The indicated metabolites from the livers of WT and DKO mice after 48 hours of fasting were measured using metabolome analysis. n = 7/group. (B) The expression of genes involved in ketogenesis was determined using quantitative real-time PCR. n = 4/group. WT vs.DKO. ⋆p<0.05, ⋆⋆p<0.01.
Figure 9.
Working model of metabolic changes in DKO mice during fasting.
(A) In WT mice, TG in adipose tissue is hydrolyzed during prolonged fasting, which releases NEFA into circulation. NEFA is taken up by various organs, including the heart, skeletal muscle and the liver as central energy substrates, which spares glucose consumption for glucose-dependent tissues, such as the brain and red blood cells. (B) However, in DKO mice, circulating NEFA cannot be efficiently taken up by the heart and skeletal muscle due to impaired FA transport via capillary ECs, which results in an increase in NEFA influx into the liver and FA accumulation in the liver. To compensate for the reduced uptake of NEFA, glucose uptake by the heart and red skeletal muscle is markedly enhanced independently of insulin even during fasting [14], which causes hypoglycemia. Although gluconeogenesis is conserved to supply glucose to peripheral tissues shortly after fasting, substrates for gluconeogenesis are reduced, resulting in insufficient gluconeogenesis during prolonged fasting, which further enhances hypoglycemia. FAO was enhanced during the fed state and a higher level of FAO was maintained even after prolonged fasting. Combined metabolic changes, including increased NEFA influx into the liver, enhanced FAO and lower blood glucose, accelerate ketogenesis. Please refer to the text and discussion for further details. Thick arrows indicate more flow; thin arrows indicate less flow; dotted arrow indicates impaired flow.
Table 2.
Primer for RT-PCR.