A Critical Role of Fatty Acid Binding Protein 4 and 5 (FABP4/5) in the Systemic Response to Fasting

During prolonged fasting, fatty acid (FA) released from adipose tissue is a major energy source for peripheral tissues, including the heart, skeletal muscle and liver. We recently showed that FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipocytes and macrophages, are prominently expressed in capillary endothelial cells in the heart and skeletal muscle. In addition, mice deficient for both FABP4 and FABP5 (FABP4/5 DKO mice) exhibited defective uptake of FA with compensatory up-regulation of glucose consumption in these tissues during fasting. Here we showed that deletion of FABP4/5 resulted in a marked perturbation of metabolism in response to prolonged fasting, including hyperketotic hypoglycemia and hepatic steatosis. Blood glucose levels were reduced, whereas the levels of non-esterified FA (NEFA) and ketone bodies were markedly increased during fasting. In addition, the uptake of the 125I-BMIPP FA analogue in the DKO livers was markedly increased after fasting. Consistent with an increased influx of NEFA into the liver, DKO mice showed marked hepatic steatosis after a 48-hr fast. Although gluconeogenesis was observed shortly after fasting, the substrates for gluconeogenesis were reduced during prolonged fasting, resulting in insufficient gluconeogenesis and enhanced hypoglycemia. These metabolic responses to prolonged fasting in DKO mice were readily reversed by re-feeding. Taken together, these data strongly suggested that a maladaptive response to fasting in DKO mice occurred as a result of an increased influx of NEFA into the liver and pronounced hypoglycemia. Together with our previous study, the metabolic consequence found in the present study is likely to be attributed to an impairment of FA uptake in the heart and skeletal muscle. Thus, our data provided evidence that peripheral uptake of FA via capillary endothelial FABP4/5 is crucial for systemic metabolism and may establish FABP4/5 as potentially novel targets for the modulation of energy homeostasis

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

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

Impaired glucose homeostasis in DKO mice during prolonged fasting.

<p>(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.</p

Primer for RT-PCR.

<p>RT PCR indicates Real Time Polymerase Chain Reaction; ACC, acetyl CoA aarboxylase; ACSL1, acyl-CoA synthetase long-chain family member 1; ApoB100, apolipoprotein B100; CPT, carnitinepalmitoyltransferase; FABP1, fatty acid binding protein 1; FAS, fatty acid synthase; FAT, fatty acid translocase; FATP2, fatty acid transport protein 2; FGF21, fibroblast growth factor 21; G6Pase, glucose-6-phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase, HMGCL, 3-hydroxymethyl-3-methylglutaryl-CoA lyase; HMGCS2, 3-hydroxy-3-methylglutaryl-CoA synthase 2; PEPCK1, phosphoenolpyruvatecarboxykinase 1, PPARα, peroxisome proliferator-activated receptor α, SCD1, stearoyl-coA desaturase 1.</p

Blood glucose is decreased while serum levels of FFA and ketone bodies are markedly increased during prolonged fasting in DKO mice.

<p>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.</p

VLDL secretion is reduced in DKO mice after fasting.

<p>(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.</p

accumulation is enhanced in DKO livers during prolonged fasting.

<p>(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.</p

Metabolome analyses of DKO livers.

<p>(nmol/g weight).</p><p>Indicated metabolites from the livers of WT and DKO mice after 48-hour of fasting were measured using capillary electrophoresis-mass spectrometry (n = 7).</p><p>Data are shown as the mean ± SE. WT vs. DKO.</p>*<p>p<0.05, **p<0.01, ***p<0.001.</p

Ketogenesis is enhanced in DKO mice.

<p>(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.</p