TGs stored in BAT in DKO mice were more depleted after cold exposure in the fasted state.

<p>The mice were exposed to a cold environment (4°C) with or without prior fasting. (A and B) TG concentration in BAT. (C and D) The total amount of TGs in interscapular BAT was calculated from the concentration of TGs in BAT and the weight of BAT. n = 4–5/group. *p<0.05; **p<0.01; ***p<0.001. (E) Gross appearance of BAT. (F) Hematoxylin/eosin staining of BAT. Bar indicates 50 µm.</p

Very low glucose levels in FABP4/5 DKO mice in the fasted state regardless of cold exposure.

<p>(A to H) Blood was collected from the retro-orbital plexus before and after cold exposure to measure the serum levels of glucose (A and B), TGs (C and D), NEFAs (E and F), and ketone bodies (G and H) in the fed (A, C, E, and G) and the fasted states (B, D, F, and H). n = 5–6/group. *p<0.05; **p<0.01; ***p<0.001. Note that the blood was collected 4 h after cold exposure for the fed groups and 2 h after cold exposure for the fasted groups because many DKO mice died during prolonged cold exposure in the fasted state.</p

Cold intolerance in FABP4/5 DKO mice with prior fasting.

<p>The cold tolerance was tested as described in the methods section. The body temperature was measured from the shaved mid-dorsal body surface. n = 5/group. *p<0.05.</p

The reduced weight of BAT in the DKO mice subjected to a 20 h fast was not altered after cold exposure.

<p>The mice were exposed to a cold environment (4°C) with or without prior fasting. (A and B) Original body weight (BW) of mice in each group before cold exposure. (A) BW of the mice included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090825#pone-0090825-g004" target="_blank">Figures 4C and 4E</a> before the experiment. (B) Left panel: BW of mice included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090825#pone-0090825-g004" target="_blank">Figures 4D and 4F</a> before fasting. Right panel: BW of mice included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090825#pone-0090825-g004" target="_blank">Figures 4D and 4F</a> after a 20 h fast but before cold exposure. (C and D) Weight of interscapular BAT in each group before and after cold exposure in the fed (C) and the fasted states (D). (E and F) The ratio of the BAT weight to the BW before fasting was calculated from the data shown above. Note that the BAT is significantly shrunk after fasting or cold exposure and that the reduced weight of BAT in the DKO mice after a 20 h fast was not altered after cold exposure. n = 4–5/group. *p<0.05; **p<0.01; ***p<0.001.</p

Reduced induction of genes associated with thermogenesis in BAT in both WT and DKO mice after cold exposure in the fasted state.

<p>The mice were maintained at room temperature or in a cold room (4°C) for 4 h with or without prior fasting. The total RNA from BAT was extracted for quantitative real-time PCR. n = 4–5/group. *p<0.05; **p<0.01; ***p<0.001.</p

Glycogen storage is depleted in SkM after a 20 h fast.

<p>The quadriceps femoris muscle was collected from mice before and after cold exposure (4°C) with or without prior fasting. (A and B) Concentration of glycogen in the quadriceps femoris muscle. n = 4–5/group. *p<0.05; ***p<0.001.</p

Uptake of ¹⁸F-FDG and ¹²⁵I-BMIPP by BAT is markedly reduced in DKO mice after cold exposure with prior fasting.

<p>(A and B) The mice received intravenous injections of ¹⁸F-FDG (100 kBq) and ¹²⁵I-BMIPP (5 kBq) via the lateral tail vein before (A) and after a 20 h fast (B). The mice were maintained at room temperature or in cold rooms (4°C) for 2 h and then sacrificed. The uptake of ¹⁸F-FDG by BAT and the uptake of ¹²⁵I-BMIPP and SkM were counted using a well-type gamma counter (n = 4–6/group). *p<0.05; **p<0.01.</p

The expression level of FABP3 and FA oxidation capacity were comparable between WT and DKO mice.

<p>(A) The mice were maintained at room temperature or in a cold room (4°C) for 4 h in the fed state. The total RNA from BAT was extracted for quantitative real-time PCR. n = 4–5/group. (B) The FA oxidation by BAT homogenates was estimated <i>in vitro</i>. The mice were placed in a cold room (4°C) for 2 h and then sacrificed. n = 4–5/group. *p<0.05; **p<0.01; ***p<0.001.</p

Working model of metabolic changes in DKO mice during fasting.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079386#pone.0079386-Iso1" target="_blank">[14]</a>, 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.</p

Fasting-induced hepatic steatosis is reversible.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079386#s4" target="_blank">Materials and Methods</a> section. n = 5−11/group. Data are shown as the mean ± SE. Control vs. no fasting/refeeding ⋆⋆p<0.01, ⋆⋆⋆p<0.001.</p