Matsuura et al Role of PHD2 in Adipocytes 2079Several cell culture studies have revealed that hypoxia and HIF convert cell metabolism that is dependent on aerobic glucose oxidation and fatty acid synthesis into that which is dependent on anaerobic glycolysis. HIF not only upregulates a series of glycolytic enzymes 15,16 but also actively inhibits oxidative phosphorylation in mitochondria by inducing pyruvate dehydrogenase kinase 1 (PDK1).17,18 PDK1 inhibits pyruvate dehydrogenase activity and consequently reduces the conversion of pyruvate to acetyl CoA, an essential substrate for oxidative phosphorylation. 17,18 In addition, HIF inhibits adipogenesis by inducing DEC1/Stra13. 13 These HIF-induced metabolic alterations such as increased glucose consumption and less fatty acid synthesis might be beneficial for nutrient excess in obese or diabetic subjects. Although HIF could be a potential therapeutic target, direct manipulation of HIF is often difficult in vivo. In contrast, PHD is an ideal target to manipulate HIF levels, and several chemical inhibitors of PHD have been developed. 19 However, the role of adipocyte PHD in the development of obesity-induced glucose intolerance has not been determined. In the present study, we generated mice lacking PHD2, also known as Egl 9 homolog1 (EglN1) in adipocytes, because PHD2 is the most crucial isoform to regulate HIF level in vitro 20 and in vivo 21 among 3 PHD isoforms (PHD1, PHD2, and PHD3). We found that PHD2 deletion in adipocyte attenuates weight gain and alleviates glucose intolerance induced by a high-fat diet (HFD). MethodsAdditional details of the experimental procedures are included in the online-only Data Supplement.All animal procedures were approved by the Animal Care and Use Committee of Kyushu University and conducted in accordance with the institutional guidelines. Previously generated Phd2-floxed mice f/f mice. Phd2 f/f mice served as controls. These mice were fed an HFD containing 60% kcal fat (High Fat Diet 32, Clea Japan, Inc) from 12 to 18 weeks of age. Mice 12 and 18 weeks of age were analyzed. Preparation of cell lysate and total RNA, Western blot analysis, quantitative reverse transcription-polymerase chain reaction, luciferase assay, and histological/immunohistochemical analysis were performed using conventional methods. The primer sequences for quantitative reverse transcription-polymerase chain reaction are shown in Table I Figure 1A and Figure IA in the online-only Data Supplement). Expression of PHD2 in heart and bone marrowderived macrophages was slightly reduced. We did not find any apparent abnormalities in the appearance in Phd2 Figure 2B and 2D). However, the extent of HFD-induced adipocyte hypertrophy was significantly reduced in Phd2 f/f /aP2-Cre mice compared with control mice (Figure 2C and 2D). A detailed analysis of the size distribution of the adipocytes revealed that WAT from controls contained a greater number of larger adipocytes (>10 000 μm 2 ) than that Enhanced Angiogenesis in WAT From HFD-Fed Phd2f/f /aP2-Cre Mice B...

Objective-Prolyl hydroxylase domain-containing proteins (PHDs) play pivotal roles in oxygen-sensing system through the regulation of ␣-subunit of hypoxia-inducible factor (HIF), a key transcription factor governing a large set of gene expression to adapt hypoxia. Although tissue hypoxia plays an essential role in maintaining inflammation, the role of PHDs in the inflammatory responses has not been clearly determined. Here, we investigated the role of PHDs in lipopolysaccharide (LPS)-induced tumor necrosis factor ␣ (TNF-␣) induction in macrophages. Methods and Results-Northern blot analysis and ELISA revealed that LPS-induced TNF-␣ upregulation was strongly suppressed by PHD inhibitors, dimethyloxallyl glycine (DMOG), and TM6008 in RAW264.7 macrophages. DMOG suppressed LPS-induced TNF-␣ upregulation in HIF-1␣-depleted cells and HIF-1␣ overexpression failed to suppress the induction of TNF-␣. DMOG rather suppressed LPS-induced NF-B transcriptional activity. Downregulation of Phd1 or Phd2 mRNA by RNA interference partially attenuated LPS-induced TNF-␣ induction. DMOG also inhibited LPS-induced TNF-␣ production in peritoneal macrophages as well as human macrophages. Conclusions-PHD inhibition by DMOG or RNA interference inhibited LPS-induced TNF-␣ upregulation in macrophages possibly through NF-B inhibition, which is independent of HIF-1␣ accumulation. This study suggests that PHDs are positive regulators of LPS-induced inflammatory process, and therefore inhibition of PHD may be a novel strategy for the treatment of inflammatory diseases. Key Words: tumor necrosis factor -alpha Ⅲ prolyl hydroxylase domain-containing protein Ⅲ hypoxia-inducible factor Ⅲ inflammation Ⅲ hypoxia I nflammation is a fundamental process for the protection of our body against outside pathogen. Tissues with inflammation are characterized by several features including the accumulation of inflammatory cells such as macrophages, lymphocytes, and neutrophils, limited blood supply attributable to impaired local microcirculation, and abnormal angiogenesis. 1 Inflammatory cells are metabolically active and consume a large amount of oxygen and nutrient. These cells are, therefore, eventually exposed to hypoxic and nutrient-deprived condition. 2 Thus, the inflammatory cells need to adapt these hypoxic conditions to perpetuate inflammatory reaction. 3 The reduced oxygen concentration is directly sensed by an innate oxygen-sensing system. 4 -6 The hypoxia-inducible factor (HIF) is a key transcription factor that mediates cellular adaptive responses to hypoxia. 7 HIF is a heterodimer consisting of an oxygen-labile ␣-subunit and a stable ␤-subunit. The stability of the ␣-subunit of HIF-1 and HIF-2 (HIF-1␣ and HIF-2␣) is regulated through the hydroxylation at the 4-position of specific proline residues in HIF-1␣ and HIF-2␣ by prolyl hydroxylase domain-containing proteins (PHDs). 8,9 Because PHD activity depends on the availability of molecular oxygen, PHDs are able to serve as a sensor for oxygen concentration. Under normal oxygen concentration, HIF-␣ i...

Human medium-chain enoyl-CoA hydratase was purified from liver, because we noticed the presence of a high medium-chain enoyl-CoA hydratase activity in human skin fibroblasts catalyzed by an enzyme different from the known enzymes catalyzing the enoyl-CoA hydratase reaction. Two enzyme preparations were obtained. One of them, preparation I, consisted of 46-kDa polypeptide, and its molecular mass was estimated to be 86 kDa. The other, preparation II, consisted of a major 77-kDa polypeptide and minor smaller polypeptides including 46-kDa polypeptide. The molecular mass of preparation II was 154 kDa. Both enzyme preparations catalyzed reversible dehydration of medium-chain D-3-hydroxyacyl-CoA to 2-trans-enoyl-CoA, but did not react with L-3-hydroxyacyl-CoA. Catalytic properties and immunochemical reactivities of these enzyme preparations were nearly the same. The cross-reactive material to the antibody was confirmed to be in peroxisomes by immunohistochemical study of cultured human skin fibroblasts.

No abstract

BackgroundHypertension induces cardiovascular hypertrophy and fibrosis. Infiltrated macrophages are critically involved in this process. We recently reported that inhibition of prolyl hydroxylase domain protein 2 (PHD2), which hydroxylates the proline residues of hypoxia‐inducible factor‐α (HIF‐α) and thereby induces HIF‐α degradation, suppressed inflammatory responses in macrophages. We examined whether myeloid‐specific Phd2 deletion affects hypertension‐induced cardiovascular remodeling.Methods and ResultsMyeloid‐specific PHD2‐deficient mice (MyPHD2KO) were generated by crossing Phd2‐floxed mice with LysM‐Cre transgenic mice, resulting in the accumulation of HIF‐1α and HIF‐2α in macrophage. Eight‐ to ten‐week‐old mice were given NG‐nitro‐L‐arginine methyl ester (L‐NAME), a nitric oxide synthase inhibitor, and Angiotensin II (Ang II) infusion. L‐NAME/Ang II comparably increased systolic blood pressure in control and MyPHD2KO mice. However, MyPHD2KO mice showed less aortic medial and adventitial thickening, and macrophage infiltration. Cardiac interstitial fibrosis and myocyte hypertrophy were also significantly ameliorated in MyPHD2KO mice. Transforming growth factor‐β and collagen expression were decreased in the aorta and heart from MyPHD2KO mice. Echocardiographic analysis showed that left ventricular hypertrophy and reduced ejection fraction induced by L‐NAME/Ang II treatment in control mice were not observed in MyPHD2KO mice. Administration of digoxin that inhibits HIF‐α synthesis to L‐NAME/Ang II‐treated MyPHD2KO mice reversed these beneficial features.ConclusionsPhd2 deletion in myeloid lineage attenuates hypertensive cardiovascular hypertrophy and fibrosis, which may be mediated by decreased inflammation‐ and fibrosis‐associated gene expression in macrophages. PHD2 in myeloid lineage plays a critical role in hypertensive cardiovascular remodeling.