Aiko Yamaguchi

Capillary Endothelial Fatty Acid Binding Proteins 4 and 5 Play a Critical Role in Fatty Acid Uptake in Heart and Skeletal Muscle

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-&bgr; 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.

Biological significance of 18F-FDG uptake on PET in patients with non-small-cell lung cancer

BACKGROUND The aim of this study is to investigate the underlying biologic mechanisms of 2-[18F]-fluoro-2-deoxy-d-glucose (18F-FDG) uptake on positron emission tomography (PET) in non-small cell lung cancer (NSCLC). METHODS One-hundred forty patients with NSCLC who underwent 18F-FDG PET were included in the study. Tumor sections were stained by immunohistochemistry for glucose transporter 1 (GLUT1), GLUT3, hypoxia-inducible factor-1 alpha (HIF-1α), hexokinase I, vascular endothelial growth factor (VEGF), microvessels (CD34), epidermal growth factor receptor (EGFR), and molecules relevant to PI3K/Akt/mTOR signaling pathway (PTEN, p-Akt, p-mTOR and p-S6). We also conducted in vitro studies of 18F-FDG uptake and mTOR inhibition in NSCLC cells. RESULTS High 18F-FDG uptake was significantly associated with poor prognosis in NSCLC patients. 18F-FDG uptake was significantly correlated with GLUT1, hexokinase I, HIF-1α, VEGF, CD34, p-Akt, p-mTOR and EGFR. PTEN expression showed inverse correlation with 18F-FDG uptake. In in vitro study, 18F-FDG uptake was markedly decreased by the inhibition of GLUT1 and GLUT1 upregulation by the induction of HIF-1α increased the 18F-FDG uptake. Inhibition of both mTOR complex1 (mTORC1) and mTORC2 suppressed cell growth, but activity of mTORC1 regulated the 18F-FDG uptake. NCI-H1650 cells with PTEN loss showed the highest 18F-FDG uptake and the least sensitivity to mTOR inhibitors. CONCLUSION The amount of 18F-FDG accumulation is associated with molecules relevant to glucose metabolism, hypoxia, angiogenesis and mTOR signaling pathway. Especially, PTEN status may affect not only 18F-FDG uptake but also effect of mTOR inhibitors on the growth of NSCLC.

Clinical significance of L-type amino acid transporter 1 expression as a prognostic marker and potential of new targeting therapy in biliary tract cancer

BackgroundThe expression of L-type amino acid transporter 1 (LAT1) has been described to play essential roles in tumor cell growth and survival. However, it remains unclear about the clinicopathological significance of LAT1 expression in biliary tract cancer. This study was conducted to determine biological significance of LAT1 expression and investigate whether LAT1 could be a prognostic biomarker for biliary tract cancer.MethodsA total of 139 consecutive patients with resected pathologic stage I-IV biliary tract adenocarcinoma were retrospectively reviewed. Tumor specimens were stained by immunohistochemistry for LAT1, Ki-67, microvessel density determined by CD34, and p53; and prognosis of patients was correlated. Biological significance of LAT1 expression was investigated by in vitro and in vivo experiments with LAT inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) using cholangiocarcinoma cell line.ResultsIn total patients, high LAT1 expressions were recognized in 64.0%. The expression of LAT1 was closely correlated with lymphatic metastases, cell proliferation and angiogenesis, and was a significant indicator for predicting poor outcome after surgery. LAT1 expression was a significant independent predictor by multivariate analysis. Both in vitro and in vivo preliminary experiments indicated that BCH significantly suppressed growth of the tumor and yielded an additive therapeutic efficacy to gemcitabine and 5-FU.ConclusionsHigh expression of LAT1 is a promising pathological marker to predict the outcome in patients with biliary tract adenocarcinoma. Inhibition of LAT1 may be an effective targeted therapy for this distressing disease.

Peroxisome Proliferator‐Activated Receptor‐γ in Capillary Endothelia Promotes Fatty Acid Uptake by Heart During Long‐Term Fasting

Background Endothelium is a crucial blood–tissue interface controlling energy supply according to organ needs. We investigated whether peroxisome proliferator‐activated receptor‐γ (PPARγ) induces expression of fatty acid–binding protein 4 (FABP4) and fatty acid translocase (FAT)/CD36 in capillary endothelial cells (ECs) to promote FA transport into the heart. Methods and Results Expression of FABP4 and CD36 was induced by the PPARγ agonist pioglitazone in human cardiac microvessel ECs (HCMECs), but not in human umbilical vein ECs. Real‐time PCR and immunohistochemistry of the heart tissue of control (Ppargfl/null) mice showed an increase in expression of FABP4 and CD36 in capillary ECs by either pioglitazone treatment or 48 hours of fasting, and these effects were not found in mice deficient in endothelial PPARγ (Pparg∆EC/null). Luciferase reporter constructs of the Fabp4 and CD36 promoters were markedly activated by pioglitazone in HCMECs through canonical PPAR‐responsive elements. Activation of PPARγ facilitated FA uptake by HCMECs, which was partially inhibited by knockdown of either FABP4 or CD36. Uptake of an FA analogue, 125I‐BMIPP, was significantly reduced in heart, red skeletal muscle, and adipose tissue in Pparg∆EC/null mice as compared with Ppargfl/null mice after olive oil loading, whereas those values were comparable between Ppargfl/null and Pparg∆EC/null null mice on standard chow and a high‐fat diet. Furthermore, Pparg∆EC/null mice displayed slower triglyceride clearance after olive oil loading. Conclusions These findings identified a novel role for capillary endothelial PPARγ as a regulator of FA handing in FA‐metabolizing organs including the heart in the postprandial state after long‐term fasting.

CD36 is indispensable for thermogenesis under conditions of fasting and cold stress

Hypothermia can occur during fasting when thermoregulatory mechanisms, involving fatty acid (FA) utilization, are disturbed. CD36/FA translocase is a membrane protein which facilitates membrane transport of long-chain FA in the FA consuming heart, skeletal muscle (SkM) and adipose tissues. It also accelerates uptake of triglyceride-rich lipoprotein by brown adipose tissue (BAT) in a cold environment. In mice deficient for CD36 (CD36(-/-) mice), FA uptake is markedly reduced with a compensatory increase in glucose uptake in the heart and SkM, resulting in lower levels of blood glucose especially during fasting. However, the role of CD36 in thermogenic activity during fasting remains to be determined. In fasted CD36(-/-) mice, body temperature drastically decreased shortly after cold exposure. The hypothermia was accompanied by a marked reduction in blood glucose and in stores of triacylglycerols in BAT and of glycogen in glycolytic SkM. Biodistribution analysis using the FA analogue (125)I-BMIPP and the glucose analogue (18)F-FDG revealed that uptake of FA and glucose was severely impaired in BAT and glycolytic SkM in cold-exposed CD36(-/-) mice. Further, induction of the genes of thermogenesis in BAT was blunted in fasted CD36(-/-) mice after cold exposure. These findings strongly suggest that CD36(-/-) mice exhibit pronounced hypothermia after fasting due to depletion of energy storage in BAT and glycolytic SkM and to reduced supply of energy substrates to these tissues. Our study underscores the importance of CD36 for nutrient homeostasis to survive potentially life-threatening challenges, such as cold and starvation.

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.

Fatty Acid Binding Protein 4 and 5 Play a Crucial Role in Thermogenesis under the Conditions of Fasting and Cold Stress

Hypothermia is rapidly induced during cold exposure when thermoregulatory mechanisms, including fatty acid (FA) utilization, are disturbed. FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipose tissues and macrophages, have been identified as key molecules in the pathogenesis of overnutrition-related diseases, such as insulin resistance and atherosclerosis. We have recently shown that FABP4/5 are prominently expressed in capillary endothelial cells in the heart and skeletal muscle and play a crucial role in FA utilization in these tissues. However, the role of FABP4/5 in thermogenesis remains to be determined. In this study, we showed that thermogenesis is severely impaired in mice lacking both FABP4 and FABP5 (DKO mice), as manifested shortly after cold exposure during fasting. In DKO mice, the storage of both triacylglycerol in brown adipose tissue (BAT) and glycogen in skeletal muscle (SkM) was nearly depleted after fasting, and a biodistribution analysis using 125I-BMIPP revealed that non-esterified FAs (NEFAs) are not efficiently taken up by BAT despite the robustly elevated levels of serum NEFAs. In addition to the severe hypoglycemia observed in DKO mice during fasting, cold exposure did not induce the uptake of glucose analogue 18F-FDG by BAT. These findings strongly suggest that DKO mice exhibit pronounced hypothermia after fasting due to the depletion of energy storage in BAT and SkM and the reduced supply of energy substrates to these tissues. In conclusion, FABP4/5 play an indispensable role in thermogenesis in BAT and SkM. Our study underscores the importance of FABP4/5 for overcoming life-threatening environments, such as cold and starvation.

Activation of pyruvate dehydrogenase by dichloroacetate has the potential to induce epigenetic remodeling in the heart

Dichloroacetate (DCA) promotes pyruvate entry into the Krebs cycle by inhibiting pyruvate dehydrogenase (PDH) kinase and thereby maintaining PDH in the active dephosphorylated state. DCA has recently gained attention as a potential metabolic-targeting therapy for heart failure but the molecular basis of the therapeutic effect of DCA in the heart remains a mystery. Once-daily oral administration of DCA alleviates pressure overload-induced left ventricular remodeling. We examined changes in the metabolic fate of pyruvate carbon (derived from glucose) entering the Krebs cycle by metabolic interventions of DCA. (13)C6-glucose pathway tracing analysis revealed that instead of being completely oxidized in the mitochondria for ATP production, DCA-mediated PDH dephosphorylation results in an increased acetyl-CoA pool both in control and pressure-overloaded hearts. DCA induces hyperacetylation of histone H3K9 and H4 in a dose-dependent manner in parallel to the dephosphorylation of PDH in cultured cardiomyocytes. DCA administration increases histone H3K9 acetylation in in vivo mouse heart. Interestingly, DCA-dependent histone acetylation was associated with an up-regulation of 2.3% of genes (545 out of 23,474 examined). Gene ontology analysis revealed that these genes are highly enriched in transcription-related categories. This evidence suggests that sustained activation of PDH by DCA results in an overproduction of acetyl-CoA, which exceeds oxidation in the Krebs cycle and results in histone acetylation. We propose that DCA-mediated PDH activation has the potential to induce epigenetic remodeling in the heart, which, at least in part, forms the molecular basis for the therapeutic effect of DCA in the heart.

Efficacy of system l amino acid transporter 1 inhibition as a therapeutic target in esophageal squamous cell carcinoma

System l amino acid transporter 1 (LAT1) is highly expressed in various types of human cancer, and contributes to cancer growth and survival. Recently, we have shown that LAT1 expression is closely related to the growth and aggressiveness of esophageal cancer, and is an independent marker of poor prognosis. However, it remains unclear whether LAT1 inhibition could suppress esophageal cancer growth. In this study, we investigated the tumor‐suppressive effects of the inhibition of LAT1. Both LAT1 and CD98, which covalently associates to LAT1 on the membrane, were expressed in human esophageal cancer cell lines KYSE30 and KYSE150. Quantitative PCR analysis showed that the expression of LAT1 was much higher than other subtypes of LAT. A selective inhibitor of LAT, 2‐aminobicyclo‐(2,2,1)‐heptane‐2‐carboxylic acid (BCH), suppressed cellular uptake of l‐14C‐leucine and cell proliferation in a dose‐dependent manner. It also suppressed phosphorylation of mammalian target of rapamycin, 4E‐BP1, and p70S6K protein, and induced cell cycle arrest at G1 phase. These results suggest that suppression of both mammalian target of rapamycin signaling and cell cycle progression is involved in BCH‐induced growth inhibition. In tumor‐bearing mice, daily treatment with BCH significantly delayed tumor growth and decreased glucose metabolism, indicating that LAT1 inhibition potentially suppresses esophageal cancer growth in vivo. Thus, our results suggest that LAT1 inhibition could be a promising molecular target for the esophageal cancer therapy.

Development of a Widely Usable Amino Acid Tracer: 76Br-α-Methyl-Phenylalanine for Tumor PET Imaging

Radiolabeled amino acids are superior PET tracers for the imaging of malignant tumors, and amino acids labeled with 76Br, an attractive positron emitter because of its relatively long half-life (16.2 h), could potentially be a widely usable tumor imaging tracer. In this study, in consideration of its stability and tumor specificity, we designed two 76Br-labeled amino acid derivatives, 2-76Br-bromo-α-methyl-l-phenylalanine (2-76Br-BAMP) and 4-76Br-bromo-α-methyl-l-phenylalanine (4-76Br-BAMP), and investigated their potential as tumor imaging agents. Methods: Both 76Br- and 77Br-labeled amino acid derivatives were prepared. We performed in vitro and in vivo stability studies and cellular uptake studies using the LS180 colon adenocarcinoma cell line. Biodistribution studies in normal mice and in LS180 tumor–bearing mice were performed, and the tumors were imaged with a small-animal PET scanner. Results: Both 77Br-BAMPs were stable in the plasma and in the murine body. Although both 77Br-BAMPs were taken up by LS180 cells and the uptake was inhibited by L-type amino acid transporter 1 inhibitors, 2-77Br-BAMP exhibited higher uptake than 4-77Br-BAMP. In the biodistribution studies, 2-77Br-BAMP showed more rapid blood clearance and lower renal accumulation than 4-77Br-BAMP. More than 90% of the injected radioactivity was excreted in the urine by 6 h after the injection of 2-77Br-BAMP. High tumor accumulation of 2-77Br-BAMP was observed in tumor-bearing mice, and PET imaging with 2-76Br-BAMP enabled clear visualization of the tumors. Conclusion: 2-77Br-BAMP exhibited preferred pharmacokinetics and high LS180 tumor accumulation, and 2-76Br-BAMP enabled clear visualization of the tumors by PET imaging. These findings suggest that 2-76Br-BAMP could constitute a potential new PET tracer for tumor imaging and may eventually enable the wider use of amino acid tracers.