Capillary endothelial fatty acid binding proteins 4 and 5 play a critical role in fatty acid uptake in heart and skeletal muscle

Abstract

Objective: Fatty acids (FAs) are the major substrate for energy production in the heart. Here, we hypothesize that capillary endothelial fatty acid binding protein 4 (FABP4) and FABP5 play an important role in providing sufficient FAs to the myocardium.

Approach and results: Both FABP4/5 were abundantly expressed in capillary endothelium in the heart and skeletal muscle. The uptake of a FA analogue, 125I-15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid, was significantly reduced in these tissues in double-knockout (DKO) mice for FABP4/5 compared with wild-type mice. In contrast, the uptake of a glucose analogue, 18F-fluorodeoxyglucose, was remarkably increased in DKO mice. The expression of transcripts for the oxidative catabolism of FAs was reduced during fasting, whereas transcripts for the glycolytic pathway were not altered in DKO hearts. Notably, metabolome analysis revealed that phosphocreatine and ADP levels were significantly lower in DKO hearts, whereas ATP content was kept at a normal level. The protein expression levels of the glucose transporter Glut4 and the phosphorylated form of phosphofructokinase-2 were increased in DKO hearts, whereas the phosphorylation of insulin receptor-β and Akt was comparable between wild-type and DKO hearts during fasting, suggesting that a dramatic increase in glucose usage during fasting is insulin independent and is at least partly attributed to the post-transcriptional and allosteric regulation of key proteins that regulate glucose uptake and glycolysis.

Conclusions: Capillary endothelial FABP4/5 are required for FA transport into FA-consuming tissues that include the heart. These findings identify FABP4/5 as promising targets for controlling the metabolism of energy substrates in FA-consuming organs that have muscle-type continuous capillary.

Keywords: capillaries; endothelial cells; fatty acid binding proteins; fatty acids; glucose; metabolism.

Figure 1

Fatty acid binding protein 4 (FABP4) and FABP5 are expressed in capillary endothelial cells. A, Expression of mRNA for Fabp4/5 in white adipose tissue (WAT) and ventricles (Vent) from wild-type (WT), Fabp4^−/−, Fabp5^−/−, and Fabp4/5 double-knockout (DKO) mice. Total RNA was isolated for semiquantitative reverse transcription polymerase chain reaction. B, Immunohistochemical localization of FABP4/5 was detected with antibodies (Ab) directed against FABP4 and FABP5 in indicated tissues from wild-type (WT), Fabp4^−/−, and Fabp5^−/− mice. Nuclei were stained with hematoxylin. Scale bar, 100 μm. See also Figure I in the online-only Data Supplement. F indicates female; M, male; and Quadri, quadriceps.

Figure 2

Glucose is a major energy substrate in the hearts and red skeletal muscles of fatty acid binding protein 4/5 (Fabp4/5) double-knockout (DKO) mice even during fasting. A and B, After a 24-hour fast, mice received intravenous injection of ¹²⁵I-15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid (¹²⁵I-BMIPP; 5 kBq) and ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG; 100 kBq) via the lateral tail vein. The animals were euthanized at 2 hours after injection. The uptake of ¹²⁵I-BMIPP (A) and ¹⁸F-FDG (B) by indicated organs from wild-type (WT) or Fabp4/5 DKO male mice was counted in a well-type gamma counter with or without 2-hour refeeding (n=6; *P<0.05). C, Representative positron emission tomography image of ¹⁸F-FDG 2 hours after injection in WT and Fabp4/5 DKO mice after a 24-hour fast. D, Baseline and insulin-stimulated uptake of FAs and glucose in isolated cardiac myocytes from WT and Fabp4/5 DKO mice. The uptake of ¹⁴C-palmitic acid (¹⁴C-PA) and ³H-deoxyglucose (³H-DG) were measured (n=6) in the presence or absence of insulin (1 mmol/L). *P<0.05. See also Figure II in the online-only Data Supplement. Bld indicates blood; D, diaphragma, Fat, gonadal fat pad; G, gastrocnemius; Hrt, heart; Liv, liver; Q, quadriceps; R-M, red skeletal muscle (soleus); S, soleus; and W-M, white skeletal muscle (quadriceps).

Figure 3

Expression of genes associated with fatty acid (FA) metabolism in hearts. A, Relative mRNA expression of genes associated with FA metabolism in the hearts of wild-type (WT) and fatty acid binding protein 4/5 (Fabp4/5) double-knockout (DKO) mice with or without a 24-hour fast. The level of each transcript is determined by real-time polymerase chain reaction (n=6). Data are shown as the mean±SD. *P<0.05. B, Triacylglycerol (TG) content in the hearts of WT and Fabp4/5 DKO mice with or without a 24-hour fast (n=5; *P<0.05). C, TG droplet is detected by oil red O stain in the hearts of WT and DKO mice with or without fasting. Scale bar, 100 μm.

Figure 4

Glucose uptake is accelerated independently of insulin signaling during fasting. A, Protein expression and phosphorylation were determined by Western blot analysis in the hearts before and after a 24-hour fast. B, Levels of protein expression or phosphorylation were quantified by densitometric analysis (n=6). Data are shown as the mean±SD. *P<0.05. C, Insulin (5 U/kg) was injected into the peritoneal cavity after a 24-hour fast. Twenty minutes later, protein was extracted from isolated hearts for Western blot analysis. D, Levels of phosphorylation were quantified by densitometric analysis (n=6). AMPK indicates AMP-activated protein kinase; AU, arbitrary unit; IRβ, insulin receptor-β; p-AMPK, phosphorylated AMPK; PFK2, phosphofructokinase-2; PDHA, pyruvate dehydrogenase-α; p-IR, phosphorylated IR; and pPFK, phophorylated PFK.

Figure 5

Metabolome analyses of fatty acid binding protein 4/5 (Fabp4/5) double-knockout (DKO) hearts. Indicated metabolites from the hearts of wild-type (WT) and Fabp4/5 DKO mice after a 24-hour fast were measured by capillary electrophoresis-mass spectrometry (n=6). *P<0.05, **P<0.01. A, Energy reserve (phosphocreatine [PCr] plus ADP) was decreased in Fabp4/5 DKO hearts. B, Metabolites in the tricarboxylic acid (TCA) cycle and malonyl-CoA as an intermediate of FA synthesis. C, The level of β-hydroxybutyrate was markedly higher in DKO hearts.

Figure 6

Serum levels of nonesterified fatty acids (NEFAs) and ketone bodies are higher in fatty acid binding protein 4/5 (Fabp4/5) double-knockout (DKO) mice after a 24-hour fast. Serum levels of indicated biochemical parameters were measured before and after a 24-hour fast (n=8). *P<0.05. TC indicates total cholesterol; and TG, triacylglycerol.