HIF Prolyl 4-Hydroxylase-2 Inhibition Improves Glucose and Lipid Metabolism and Protects Against Obesity and Metabolic Dysfunction

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1 3324 Diabetes Volume 63, October 2014 Lea Rahtu-Korpela, 1 Sara Karsikas, 1 Sohvi Hörkkö, 2,3 Roberto Blanco Sequeiros, 4 Eveliina Lammentausta, 4 Kari A. Mäkelä, 5 Karl-Heinz Herzig, 5 Gail Walkinshaw, 6 Kari I. Kivirikko, 1 Johanna Myllyharju, 1 Raisa Serpi, 1 and Peppi Koivunen 1 HIF Prolyl 4-Hydroxylase-2 Inhibition Improves Glucose and Lipid Metabolism and Protects Against Obesity and Metabolic Dysfunction Diabetes 2014;63: DOI: /db OBESITY STUDIES Obesity is a major public health problem, predisposing subjects to metabolic syndrome, type 2 diabetes, and cardiovascular diseases. Specific prolyl 4-hydroxylases (P4Hs) regulate the stability of the hypoxia-inducible factor (HIF), a potent governor of metabolism, with isoenzyme 2 being the main regulator. We investigated whether HIF-P4H-2 inhibition could be used to treat obesity and its consequences. Hif-p4h- 2 deficient mice, whether fed normal chow or a highfat diet, had less adipose tissue, smaller adipocytes, and less adipose tissue inflammation than their littermates. They also had improved glucose tolerance and insulin sensitivity. Furthermore, the mrna levels of the HIF-1 targets glucose transporters, glycolytic enzymes, and pyruvate dehydrogenase kinase-1 were increased in their tissues, whereas acetyl-coa concentration was decreased. The hepatic mrna level of the HIF-2 target insulin receptor substrate-2 was higher, whereas that of two key enzymes of fatty acid synthesis was lower. Serum cholesterol levels and de novo lipid synthesis were decreased, and the mice were protected against hepatic steatosis. Oral administration of an HIF-P4H inhibitor, FG-4497, to wild-type mice with metabolic dysfunction phenocopied these beneficial effects. HIF-P4H-2 inhibition may be a novel therapy that not only protects against the development of obesity and its consequences but also reverses these conditions. Hypoxia-inducible factor (HIF) regulates the expression of numerous hypoxia-regulated genes (1 3). The HIF-a subunit isoforms HIF-1a and HIF-2a are synthesized constitutively, and hydroxylation of two critical prolines generates 4-hydroxyproline residues that target HIF-a for degradation in normoxia. In hypoxia, this hydroxylation is inhibited so that HIF-a evades degradation and forms a functional dimer with HIF-b (1 3). The hydroxylation of HIF-a is catalyzed by HIF prolyl 4-hydroxylase (P4H) isoenzymes 1 3 (also known as PHDs 1 3 and EglNs 2, 1, and 3) (1 6) and a transmembrane, P4H-TM (3,7), with HIF-P4H-2 being the main oxygen sensor in the HIF pathway (1 3). Hif-p4h-2 null mice die during embryonic development, whereas Hif-p4h-1 and Hif-p4h-3 null mice are viable (8). Broad-spectrum conditional Hif-p4h-2 inactivation leads to severe erythrocytosis, hyperactive angiogenesis, and dilated cardiomyopathy (3,9,10). We have generated Hif-p4h-2 hypomorphic mice (Hif-p4h-2 gt/gt ) that express decreased amounts of wild-type Hif-p4h-2 mrna and show stabilization of Hifas (11). These mice appear healthy and have a normal life 1 Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research, University of Oulu, FIN Oulu, Finland 2 Nordlab Oulu, Oulu University Hospital, FIN Oulu, Finland 3 Department of Medical Microbiology and Immunology, Medical Research Center, University of Oulu, FIN Oulu, Finland 4 Department of Radiology, Oulu University Hospital and University of Oulu, FIN Oulu, Finland 5 Biocenter Oulu, Department of Physiology, University of Oulu, FIN Oulu, Finland 6 FibroGen, Inc., San Francisco, CA Corresponding author: Peppi Koivunen, peppi.koivunen@oulu.fi. Received 21 March 2014 and accepted 24 April This article contains Supplementary Data online at by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

2 diabetes.diabetesjournals.org Rahtu-Korpela and Associates 3325 span. They have no increased erythrocytosis and show no signs of hyperactive angiogenesis or dilated cardiomyopathy; instead, they are protected against myocardial infarction and ischemia-reperfusion injury (11,12). Many studies have demonstrated that hypoxia reduces body weight (13 15). The only obvious abnormality found in the Hif-p4h-2 gt/gt mice was that their weights were 85 90% of those of the wild type (11). We focused initially on this difference and found that these mice have less adipose tissue than their littermates. This finding prompted us to study their lipid and glucose metabolism, especially because obesity is a major public health problem and increases the risk of metabolic syndrome, type 2 diabetes, and cardiovascular diseases. The data show that the Hifp4h-2 gt/gt mice, whether fed normal chow or a high-fat diet (HFD), have major alterations in their adipose tissues and metabolism, including improved glucose tolerance and insulin sensitivity, reduced serum cholesterol levels, and protection against hepatic steatosis. Small-molecule HIF-P4H inhibitors have been developed for the treatment of anemias and ischemic diseases, for example (3). Our Hif-p4h-2 gt/gt mouse data suggest that pharmacological HIF-P4H-2 inhibition could also be beneficial for the treatment of obesity and metabolic syndrome. We therefore administered FG-4497 to wild-type mice with a metabolic dysfunction, which stabilizes HIF-a in cultured cells and in vivo and increases erythropoiesis in animals with no apparent toxicity (3,16,17). The data demonstrate that FG-4497 administration phenocopied the beneficial effects of genetic HIF-P4H-2 deficiency on adipose tissues and metabolism. RESEARCH DESIGN AND METHODS Animal Experiments Generation of C57BL/6 Hif-p4h-2 gt/gt mice has been described (11). All experiments were performed according to protocols approved by the Provincial State Office of Southern Finland. All data obtained with the Hif-p4h- 2 gt/gt mice were compared with those of their wild-type littermates. The mice were fed a standard rodent diet or HFD (18% and 42% kcal fat, respectively) (Teklad T.2018C.12 and TD88137; Harlan Laboratories). FG was dissolved in 0.5% sodium carboxymethyl cellulose (Spectrum) and 0.1% polysorbate 80 (Fluka), and the solvent was also used as a vehicle. Both were administered orally. Histological Analyses Five-micrometer sections of formaldehyde-fixed paraffinembedded tissue samples were stained with hematoxylineosin (H-E) and viewed and photographed with a Leica DM LB2 microscope and Leica DFC320 camera or Nikon Eclipse 50i microscope and DS-5M-L2 camera. Representative pictures (five to eight per mouse) were taken, and areas of 100 adipocytes were quantified with Nikon NIS- Elements BR 2.30 imaging software. Macrophage infiltration was analyzed by an anti-cd68 antibody (ab955; Abcam) and EnVision Detection System (Dako). The number of macrophage aggregates was calculated from five to eight fields per sample. Hepatic steatosis was scored (0 to ++++) from H-E stained sections. Western Blotting NE-PER extraction reagents (Thermo Fisher Scientific) were used to prepare nuclear fractions. Samples of mg were resolved by SDS-PAGE, blotted, and probed with the following primary antibodies: Hif-1a (NB ; Novus Biologicals), Hif-2a (ab199; Abcam), and b-actin (NB ; Novus Biologicals). Quantitative PCR Analyses Total RNA from tissues was isolated with E.Z.N.A. Total RNA Kit II (Omega Bio-Tek) or TriPure Isolation Reagent (Roche Applied Science) and reverse transcripted with an iscript cdna Synthesis Kit (Bio-Rad). Quantitative PCR was performed with itaq SYBR Green Supermix with ROX (Bio-Rad) in a Stratagene Mx3005 thermocycler or C1000 Touch Thermal Cycler and CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with the primers shown in Supplementary Table 1. MRI Analysis Tissue fat content was determined using the 3-point Dixon method and a 3-T clinical scanner (Skyra; Siemens, Erlangen, the Netherlands). The sequence used was T1 turbo spin echo Dixon (field of view ; repetition time 720; echo time 20, 24 slices). The torso of the animal was scanned, and the region of interest was drawn to separate the regional volume of subcutaneous fat for possible correlation with other variables. Glucose and Insulin Tolerance Tests and Deoxyglucose Uptake Test Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed on mice fasted for 6 12 h and anesthetized with fentanyl/fluanisone and midazolam. For GTT, mice were injected intraperitoneally with 1 mg/g glucose, and blood glucose concentrations were monitored with a glucometer. Serum insulin values were determined with Rat/Mouse Insulin ELISA kit (EZRMI- 13K; Millipore), and HOMA-IR scores were calculated from the glucose and insulin values. For ITT, mice were injected intraperitoneally with 1 IU/kg insulin (Humulin Regular; Lilly), and blood glucose concentrations were determined as for GTT. For deoxyglucose uptake test, mice were injected intraperitoneally with 0.6 mci/g 14 C-deoxyglucose (PerkinElmer) combined with 1 mg/g glucose and killed 60 min later. Fifty-microgram pieces of tissues and 50-mL blood samples were homogenized in 1:1 (or in the case of white adipose tissue [WAT], 2:1) chloroform:methanol and centrifuged. The pellet was re-extracted, and the supernatants were combined and scintillated for 14 C activity. Determination of Serum Lipids Serum total cholesterol, HDL cholesterol, and triglyceride levels were determined by an enzymatic method (Roche

3 3326 HIF-P4H-2 in Obesity and Metabolism Diabetes Volume 63, October 2014 Diagnostics). The Friedewald equation (18) was used to calculate LDL + VLDL cholesterol concentrations. Determination of Tissue Acetyl-CoA Concentration Snap-frozen tissues were homogenized in precooled 8% perchloric acid in 40% (volume for volume) ethanol and centrifuged. The supernatant was neutralized, and the amount of acetyl-coa was measured with a PicoProbe Acetyl CoA Assay Kit (ab87546; Abcam). Lipogenesis Analysis Mice were terminally anesthetized with fentanyl/fluanisone and midazolam and ;30-mg pieces of WAT and liver were excised and incubated for 90 min at 37 C with 40 mci [ 14 C]acetate (PerkinElmer) in DMEM pregassed with 95% O 2 and 5% CO 2. Lipids were extracted after saponification and scintillated for 14 C activity (19). Statistical Analyses Student two-tailed t test was used for comparisons between groups, and Student t test for paired samples was used for pairwise testing. Fisher exact test was used to calculate significance for the difference between genotypes in liver steatosis analyses. Areas under the curve were calculated by the summary measures method. All data are mean 6 SEM. P, 0.05 was considered statistically significant. RESULTS Hif-p4h-2 gt/gt Mice Are Lighter Than Their Littermates Despite No Alterations in Food Intake and Physical Activity Hif-p4h-2 gt/gt mice have their Hif-p4h-2 gene disrupted by a gene trap insertion cassette. Small amounts of wild-type Hif-p4h-2 mrna were nevertheless generated from the gene-trapped alleles (11), its smallest relative amounts in the Hif-p4h-2 gt/gt tissues studied being found in the heart and skeletal muscle, whereas the highest proportion was found in the liver (11). We analyzed these values in the skeletal muscle and liver of the mice in the present study and found that the relative proportions of wild-type Hif-p4h-2 mrna in these Hif-p4h-2 gt/gt tissues were ;25% and ;60%, respectively (Supplementary Fig. 1A). We found previously that the amount of Hif-p4h-2 protein in Hif-p4h-2 gt/gt skeletal muscle, heart, and kidney corresponds to that of the wild-type mrna (11). Hif-1a was well stabilized in the heart and weakly stabilized in the skeletal muscle and kidney of the Hif-p4h-2 gt/gt mice, whereas Hif-2a was weakly stabilized in the heart and skeletal muscle (11). We also found a weak stabilization of Hif-2a, but not of Hif-1a, in the Hif-p4h-2 gt/gt liver with a more sensitive antibody (Fig. 1A). The weight of 5-week-old Hif-p4h-2 gt/gt mice fed normal chow (18% kcal fat) was lower than that of their wildtype littermates, and the Hif-p4h-2 gt/gt mice gained less weight during the subsequent 10 weeks (Fig. 1B). The weight difference persisted later, with the weight of 1- year-old Hif-p4h-2 gt/gt mice being 80 85% of that of wild type (Fig. 1B). No difference was found between the genotypes in tibia length or liver weight relative to tibia length (Supplementary Fig. 1B). No difference was found in the amount of food intake between the Hif-p4h-2 gt/gt and wild-type mice in metabolic home cage analyses whether expressed per mouse or body weight, and no difference was found in the physical activity between these mice (Supplementary Fig. 2). There was a trend for increased O 2 consumption and CO 2 production when expressed per body weight in the Hif-p4h- 2 gt/gt mice, but this reached statistical significance only in the case of O 2 consumption in the dark, with no difference in respiratory exchange ratio between the genotypes (Supplementary Fig. 2). Hif-p4h-2 gt/gt Mice Have Less Adipose Tissue and Smaller Adipocytes The amount of gonadal WAT in the Hif-p4h-2 gt/gt mice, expressed relative to tibia length to correct for body weight differences, was one-half of that in the wild-type littermates (Fig. 1C). Furthermore, the adipocytes in the Hif-p4h-2 gt/gt WAT were smaller (Fig. 1D) and the amount of wild-type Hif-p4h-2 mrna in the Hif-p4h-2 gt/gt WAT was ;50% of that in wild-type tissue (Supplementary Fig. 1A). The amount of Hif-p4h-2 protein in the Hifp4h-2 gt/gt WAT was correspondingly reduced (Supplementary Fig. 1C), and its Hif-1a but not Hif-2a was stabilized to a low extent (Fig. 1A). The weight of the brown adipose tissue (BAT) was likewise reduced (Fig. 1E), and the wildtype Hif-p4h-2 mrna level in the Hif-p4h-2 gt/gt BAT was only ;10% of that in the wild-type tissue (Supplementary Fig. 1A). No difference in the mrna level of uncoupling protein 1 (Ucp1) was found between the Hif-p4h-2 gt/gt and wild-type BAT (Supplementary Fig. 1D). MRI analyses of live anesthetized mice indicated that the amount of subcutaneous fat was also reduced in the Hif-p4h-2 gt/gt mice (Fig. 1F). Hif-p4h-2 gt/gt Mice Have a Reduced Number of Adipose Tissue Macrophage Aggregates Obesity is associated with chronic adipose tissue inflammation and formation of macrophage aggregates around adipocytes (20,21). Chronic 21-day hypoxia decreases the number of such aggregates in 1-year-old mice (15). We found in the present study that their number is significantly lower in the WAT of 1-year-old Hifp4h-2 gt/gt mice than in wild-type mice (Fig. 1G). As in the mice subjected to chronic hypoxia (15), increased adiponectin mrna level and decreased leptin and chemochine (C-C motif) ligand 2 mrna levels were found in the Hifp4h-2 gt/gt WAT (Supplementary Fig. 3A), whereas no increase was seen in serum adiponectin level, but serum leptin level was decreased (Supplementary Fig. 3B). Hif-p4h-2 gt/gt Mice Have Reduced Serum Cholesterol Levels and Are Protected Against Hepatic Steatosis The levels of serum total cholesterol and HDL and LDL + VLDL cholesterol were lower in the Hif-p4h-2 gt/gt mice than in the wild-type mice, with the HDL/LDL + VLDL ratio being increased, whereas the triglyceride level was

4 diabetes.diabetesjournals.org Rahtu-Korpela and Associates 3327 Figure 1 Hif-p4h-2 gt/gt mice are lighter than their wild-type (wt) littermates and have less adipose tissue, smaller adipocytes, and a reduced number of macrophage aggregates in WAT. A: Western blot analyses of Hif-1a and Hif-2a protein in nuclear fractions of liver and WAT. b-actin was used as a loading control. B: Weight of 5-week-old female wt and Hif-p4h-2 gt/gt (gt/gt) mice fed normal chow and their weight gain during the subsequent 10 weeks and weight of 1-year-old female and male mice. C: Weight of gonadal WAT in 1-yearold male mice. D: Cross-sectional area of adipocytes in the WAT of 1-year-old male mice. Scale bar = 100 mm. E: Weightofsubcutaneous BAT relative to tibia length in 1-year-old female mice. F: MRI analyses of the amount of subcutaneous adipose tissue in 4-month-old female mice. G: Number of macrophage aggregates in gonadal WAT of 1-year-old male wt and gt/gt mice. Adipocytes surrounded by macrophage aggregates (*). Scale bar = 100 mm. Data are mean 6 SEM (n =4 10 per group). *P < 0.05, **P < 0.01, ***P = S.c., subcutaneous. not altered significantly (Fig. 2A). Livers of 1-year-old wild-type mice had steatosis, even though these mice were not fed an HFD, whereas the Hif-p4h-2 gt/gt mice were protected against its development (Fig. 2B). Hif-p4h-2 gt/gt Mice Have Improved Glucose Tolerance and Insulin Sensitivity The glucose tolerance of 1-year-old Hif-p4h-2 gt/gt mice was markedly better than that of their littermates, and even the fasting blood glucose levels (at 0 min) were significantly lower than in the wild type (Fig. 3A and Supplementary Fig. 4A). The fasting serum insulin levels and HOMA-IR scores were likewise lower, indicating that the lower blood glucose levels were due to increased insulin sensitivity (Fig. 3B and Supplementary Fig. 4B). A similar, though less marked, difference in glucose tolerance was seen between the genotypes in 4 5-month-old mice (Fig. 3A). Correspondingly, 3 4-month-old Hif-p4h-2 gt/gt mice had lower blood glucose levels than wild-type mice in an ITT (Fig. 3C). To learn which Hif-p4h-2 gt/gt tissues were responsible for the increased glucose intake, we studied the uptake of 14 C-deoxyglucose in fasting mice and found an increased uptake relative to the wild type in the skeletal muscle (Fig. 3D). Wild-Type But Not Hif-p4h-2 gt/gt Mice Develop Metabolic Dysfunction With Age A comparison of data between 1-year-old and 4 5-monthold wild-type mice indicated that the former had larger adipocytes, a larger number of adipose tissue macrophage aggregates, increased fasting blood glucose and serum insulin levels, and higher HOMA-IR scores, whereas none of these were significantly different between 1-year-old and 4 5-month-old Hif-p4h-2 gt/gt mice (Fig. 4A E). The 1-yearold wild-type mice thus had relative insulin resistance and metabolic dysfunction compared with the younger mice, whereas the Hif-p4h-2 gt/gt mice were protected against their development. Hif-p4h-2 gt/gt Mice Have Altered Expression of Glucose and Lipid Metabolism Genes The mrna levels of the HIF-1a targets Glut1 and several enzymes of glycolysis (22) were increased in the Hif-p4h- 2 gt/gt skeletal muscle and WAT (Fig. 5A and B) butnotin Hif-p4h-2 gt/gt liver (Supplementary Fig. 5A). The mrna level of the glucose-regulated Glut2 (23) was lower in the liver of the Hif-p4h-2 gt/gt mice than in wild-type mice, presumably because of their improved glucose tolerance, whereas the mrna level of the insulin-regulated

5 3328 HIF-P4H-2 in Obesity and Metabolism Diabetes Volume 63, October 2014 Figure 2 Hif-p4h-2 gt/gt mice have decreased serum cholesterol levels and are protected against hepatic steatosis. A: Serum total cholesterol, HDL cholesterol, and LDL + VLDL cholesterol levels; HDL/LDL + VLDL cholesterol ratios; and triglyceride (TG) levels of 7 11-month-old male wild-type (wt) and Hif-p4h-2 gt/gt (gt/gt) mice after fasting for 2h(n = 19 for wt and n = 11 for gt/gt for total cholesterol, HDL cholesterol, and TG values and n = 14 for wt and n = 5 for gt/gt for LDL + VLDL and HDL/LDL + VLDL cholesterol values). Data are mean 6 SEM. B: H-E stained liver sections of 1- year-old male mice (n = 7 for both groups). Scoring of steatosis is shown. Scale bar = 200 mm. *P < 0.05, **P = 0.005, ***P = s, serum. Glut4 (24) was higher in the skeletal muscle and WAT of the Hif-p4h-2 gt/gt mice, probably because of their increased insulin sensitivity (Fig. 5A and B). The mrna level of the HIF-1a target Pdk1, which inhibits pyruvate dehydrogenase activity (22), was increased in the Hifp4h-2 gt/gt skeletal muscle and WAT (Fig. 5A). The Pparg mrna level was likewise increased in the Hif-p4h-2 gt/gt skeletal muscle and WAT (Fig. 5A), this change being similar to that seen in the WAT of mice with adipocytespecific Hif-p4h-2 deletion (25), whereas the Ppara mrna level was slightly decreased in the Hif-p4h-2 gt/gt liver (Fig. 5B). The mrna levels of the lipolysis markers Lipe and Pnpla2 were increased in the Hif-p4h-2 gt/gt WAT (Fig. 5A), suggesting that an increased lipolysis may have contributed to the decreased amount of WAT in these mice. The mrna levels of Srebp1c, which regulates lipogenesis and fatty acid synthesis, and its targets Acca and Fas, enzymes of fatty acid synthesis, were lower in the Hif-p4h-2 gt/gt liver, whereas the mrna level of the Ldl receptor was similar in the Hif-p4h-2 gt/gt and wild-type livers (Fig. 5B). The mrna level of the HIF-2a target Irs2 (26), which regulates Srepb1c and hepatic lipid accumulation (27), was increased in the Hif-p4h-2 gt/gt liver, whereas the mrna level of Irs1 was not altered (Fig. 5B). To study whether the increased mrna levels led to increased protein levels, we analyzed Glut4, Gadph, and Pdk1 in WAT by Western blotting and found increased levels of all three proteins in the Hif-p4h-2 gt/gt WAT (Supplementary Fig. 5B). Hif-p4h-2 gt/gt Mice Have Reduced Acetyl-CoA Levels and De Novo Lipogenesis To study whether the presumed decreased conversion of pyruvate to acetyl-coa as a result of an increased Pdk1 expression actually decreased the amount of acetyl-coa, we measured its amount in skeletal muscle, WAT, and liver and found a decreased concentration in all three Hifp4h-2 gt/gt tissues (Fig. 5C). We also studied whether the decreased Acca and Fas mrna and acetyl-coa levels decreased de novo lipogenesis by incubating fresh tissue slices with [ 14 C]acetate and measuring the incorporation of radioactivity into extractable lipids and found decreased lipogenesis in the Hif-p4h-2 gt/gt WAT (P = 0.03) and liver (P = 0.06) (Fig. 5D). Hif-p4h-2 gt/gt Mice Are Protected Against HFD- Induced Metabolic Changes and Steatosis To study whether Hif-p4h-2 gt/gt mice are protected against obesity-induced changes in glucose metabolism, 6-monthold mice were fed an HFD (42% kcal fat) for 6 weeks. The weight gain of the Hif-p4h-2 gt/gt and wild-type mice during the HFD treatment was similar, the Hif-p4h-2 gt/gt mice thus remaining lighter than their littermates (Fig. 6A). The adipocytes were smaller and the number of macrophage aggregates lower in the Hif-p4h-2 gt/gt WAT than in the wild-type WAT (Fig. 6B and C and Supplementary Fig. 6A). The glucose tolerance of the HFD-fed Hif-p4h-2 gt/gt mice was better than that of the HFD-fed wild-type mice, and their fasting blood glucose levels (at 0 min) were likewise lower (Fig. 6D), whereas the lower serum insulin values (by 15%) and HOMA-IR scores (by 36%) in the HFD-fed Hif-p4h-2 gt/gt mice were not statistically significant (Supplementary Fig. 6B). Livers of all 6-month-old HFD-treated wild-type mice had steatosis (Fig. 6E), with four of the nine having a steatosis score of ++++, whereas only four of the eight Hif-p4h-2 gt/gt mice had steatosis (P = 0.03), with only one of these having a score of +++ and none having Thus, the Hif-p4h-2 gt/gt mice were protected against the development of HFD-induced hepatic steatosis. Pharmacological Hif-p4h Inhibition Reverses Metabolic Dysfunction Both in Aged Wild-Type Mice and in Mice Fed an HFD FG-4497 inhibits all three HIF-P4Hs competitively with respect to 2-oxoglutarate, with similar IC 50 values (7). We studied whether its oral administration can be used to reverse metabolic dysfunction in two models: 1) 1- year-old wild-type mice fed normal chow that were shown to have metabolic dysfunction (Fig. 4) and 2) 3.5-month-old wild-type mice fed HFD for 6 weeks before the administration of 60 mg/kg FG-4497 on days 1, 3, and 5 of each week was begun (7). This FG-4497 dose stabilizes Hif-1a and Hif-2a in mouse kidney and liver and increases serum erythropoietin concentration about sixfold (7). After a 1-week adjustment period, FG-4497 administration to 1-year-old mice fed normal chow reduced the

6 diabetes.diabetesjournals.org Rahtu-Korpela and Associates 3329 Figure 3 Hif-p4h-2 gt/gt (gt/gt) mice have improved glucose tolerance, insulin sensitivity, and increased deoxyglucose uptake into skeletal muscle. A: GTTs of 1-year-old and 4 5-month-old female wild-type (wt) and gt/gt mice. The 0-min value was determined after fasting for 12h(n =9 17 per group). B: Serum insulin levels and HOMA-IR scores determined from the GTT of the 1-year-old mice in A. C: ITT of 3 4- month-old female mice. The 0-min value was determined after fasting for 6 h (n =5 6 per group). Data are relative to glucose values at 0 min. D: Deoxyglucose uptake test. Eight sibling pairs of wt and gt/gt mice were fasted for 12 h. 14 C-deoxyglucose was then injected intraperitoneally, the mice were killed 60 min later, and their tissues were homogenized and analyzed for radioactivity. Disintegrations per min per milligram for each tissue were compared between the wt and gt/gt members of each sibling pair. Data are mean 6 SEM. *P < 0.05, **P < 0.01.b,blood;s,serum. weight of these mice during the subsequent 5 weeks by ;1.3 g, whereas the vehicle-treated mice gained ;0.6 g (Fig. 7A). The adipocytes were smaller and the number of macrophage aggregates lower in the WAT of the FG treated than those of the vehicle-treated mice (Fig. 7B and C). The serum total cholesterol level and the HDL and LDL + VLDL cholesterol levels of the FG treated mice were significantly decreased, whereas Figure 4 One-year-old wild-type (wt) but not Hif-p4h-2 gt/gt (gt/gt) mice showed metabolic dysfunction relative to corresponding younger mice. A: Cross-sectional area of adipocytes. B: Number of macrophage aggregates. C: Fasting (12-h) blood glucose values. D: Fasting (12-h) serum insulin values. E: HOMA-IR scores in 4 5-month-old (young [y]) and 1-year-old (old [o]) wt and gt/gt mice (n =5 14 per group). Data are mean 6 SEM. *P < 0.05, **P < 0.01, ***P < b, blood; s, serum.

7 3330 HIF-P4H-2 in Obesity and Metabolism Diabetes Volume 63, October 2014 Figure 5 Hif-p4h-2 gt/gt (gt/gt) mice have altered expression of genes of glucose and lipid metabolism and reduced acetyl-coa levels and show reduced lipogenesis. A and B: Quantitative PCR analyses of the mrna levels of HIF target and lipid metabolism genes in the skeletal muscle and WAT (A) and liver (B) of gt/gt mice relative to the wild type (wt). Acca, acetyl-coa carboxylase a; ApoB, apolipoprotein B; Fas, fatty acid synthase; Hk1, hexokinase-1; Irs1 and Irs2, insulin receptor substrates 1 and 2; Ldha, lactate dehydrogenase a; Ldlr, LDL receptor; Lipe, hormone-sensitive lipase; Pdk1 and Pdk4, pyruvate dehydrogenase kinases 1 and 4; Pfkl, phosphofructokinase; Pgk1, phosphoglycerate kinase-1; Pnpla2, patatin-like phospholipase domain-containing protein 2; Pparg and Ppara, peroxisome proliferator activated receptors g and a; Srebp1c, sterol regulatory element binding protein 1c. The expression of each gene was studied relative to b-actin (n =6 9 per group). C: Level of acetyl-coa in the skeletal muscle, WAT, and liver. D: Incorporation of radioactivity from [ 14 C]acetate into extractable lipids as an indication of lipogenesis in the WAT and liver of 4 5-month-old male wt and gt/gt mice, expressed per milligram tissue wet weight (n =4 7 per group in C and n =9 10 per group in D). Data are mean 6 SEM. *P < 0.05, **P < 0.01, ***P < their HDL/LDL + VLDL ratio was increased (Fig. 7D). The fasting blood glucose levels of the FG-4497 treated mice were likewise decreased, whereas the decreases in the serum insulin level by ;25% and HOMA-IR score by ;75% (Fig. 7E) were not statistically significant (P = 0.11 in both cases). In the other model, 2-month-old mice were fed normal chow or HFD for 6 weeks, after which the mice fed Figure 6 Hif-p4h-2 gt/gt (gt/gt) mice are protected against HFD-induced metabolic changes and steatosis. A: Weight of 6-month-old female wild-type (wt) and gt/gt mice before and after administration of HFD for 6 weeks (n = 7 10 per group). B: Cross-sectional area of WAT adipocytes. C: Number of macrophage aggregates (aggregates/field) in gonadal WAT. D: GTT after the 6-week HFD administration. The 0-min value was determined after fasting for 12 h. E: H-E stained liver sections of these HFD-fed mice. Scoring of steatosis is shown. Scale bar = 200 mm. Data are mean 6 SEM. *P < 0.05, **P < b, blood.

8 diabetes.diabetesjournals.org Rahtu-Korpela and Associates 3331 Figure 7 Pharmacological Hif-p4h inhibition reverses metabolic dysfunction in both aged mice and mice fed HFD. A E: One-year-old male wild-type mice fed normal chow (NC) were given vehicle or 60 mg/kg of FG-4497 on days 1, 3, and 5 of each week for 6 weeks (n =6 7 per group). A: Weight gain of the mice at 6 weeks relative to their weights at 1 week (adjustment period). B: Cross-sectional area of WAT adipocytes. Scale bar = 100 mm. C: Number of WAT macrophage aggregates (*) (aggregates/field). Scale bar = 100 mm. D: Serum total cholesterol, HDL cholesterol, and LDL + VLDL cholesterol levels and HDL/LDL + VLDL cholesterol ratios measured after fasting for 2 h. E: Blood glucose and serum insulin levels and HOMA-IR scores determined from the samples in D. F I: Two-month-old male wild-type mice (n =8 10 per group) were fed NC or HFD (42% kcal fat) for 6 weeks, after which the mice fed NC were given vehicle and those fed HFD either HFD and vehicle or HFD and FG-4497 for 4 weeks as in A E. F: Weight of the mice fed NC or HFD before and after the administration of vehicle or FG G: Weight of gonadal WAT of HFD-fed mice after the 4-week vehicle or FG-4497 treatment. H: GTT of the HFD-fed mice after the 4-week vehicle or FG-4497 administration. The 0-min value was determined after fasting for 12 h. I: Serum insulin levels and HOMA-IR scores determined from the GTT in H. Data are mean 6 SEM. *P < 0.05, **P < 0.01, ***P < b, blood; s, serum; Veh, vehicle. normal chow were given vehicle, and those fed HFD were given either HFD and vehicle or HFD and FG-4497 for 4 weeks. During the initial 6-week period, the HFD-fed mice gained more weight than those fed normal chow (Fig. 7F). The FG-4497 treatment decreased the weight of the HFD-fed mice by ;0.6 g, whereas their vehicletreated controls gained ;3.0 g (FG-4497 vs. vehicle mice P =0.02)(Fig.7F).TheWATweightoftheFG-4497 treated HFD mice was lower than that of their controls (Fig. 7G). The glucose tolerance of the FG-4497 treated HFD mice was better than that of their vehicle-treated controls (Fig. 7H), and their fasting serum insulin levels and HOMA-IR scores were significantly decreased (Fig. 7I).

9 3332 HIF-P4H-2 in Obesity and Metabolism Diabetes Volume 63, October 2014 DISCUSSION The data indicate that Hif-p4h-2 gt/gt mice fed either a normal chow or an HFD have less adipose tissue, smaller adipocytes, a decreased number of adipose tissue macrophage aggregates, and lower serum cholesterol levels than their littermates. They are also protected against hepatic steatosis and show increased glucose tolerance and insulin sensitivity. The Hif-p4h-2 gt/gt mice had higher levels of glucose transporters and glycolysis enzymes in their skeletal muscle and WAT, with similar higher levels previously found in their hearts (11). Uptake of deoxyglucose in skeletal muscle was also increased. Furthermore, Pdk1 expression was increased in the Hif-p4h-2 gt/gt skeletal muscle and WAT, as has also been found in the Hif-p4h-2 gt/gt heart (11). A higher Pdk1 level may further increase glycolysis by inhibiting the entry of pyruvate into the citric acid cycle (22). Thus, it appears that glycolysis is increased in several Hif-p4h-2 gt/gt tissues, contributing to the overall improved glucose tolerance (Supplementary Fig. 7). These changes agree with the established consequences of the stabilization of HIF-1a (22). HIF-P4H-1 and -3 have been reported to also have enzyme-specific substrates other than HIF-1a and HIF-2a (1 3), and thus, changes in the levels of those two enzymes may also influence HIFindependent pathways. Such substrates have so far not been identified for HIF-P4H-2; therefore, it seems likely that most, if not all, of the metabolic changes found in the Hif-p4h-2 gt/gt mice were mediated by Hif-a. Obesity is associated with a chronic low-grade inflammation that predisposes to insulin resistance. Adipose tissue macrophages are believed to play a key role in obesity-induced insulin resistance (20,21). They infiltrate obese adipose tissue and along with the hypertrophied adipocytes, release cytokines and adipokines that contribute to the proinflammatory response (20). Macrophagederived proinflammatory factors block insulin action in adipocytes by downregulating the expression of the insulinregulated GLUT4 and impairing insulin-stimulated GLUT4 transport to the plasma membrane (20). Because we found decreased size of adipocytes, a reduced number of adipose tissue macrophages, and increased Glut4 expression in the Hif-p4h-2 gt/gt mice, it seems likely that these changes contribute to increased insulin sensitivity in these mice (Supplementary Fig. 7). Increased expression of the Hif-2a target Irs2 in the liver of mice with acute hepatic Hif-p4h-3 deletion was accompanied by a decreased Srebp1c expression (26). The present results indicating weak stabilization of Hif- 2a, increased expression of Irs2, and decreased expression of Srebp1c and its targets Acca and Fas in the Hif-p4h-2 gt/gt liver agree with those data. These changes are probably responsible for the decreased fatty acid synthesis and de novo lipogenesis found in the Hif-p4h-2 gt/gt liver and WAT. The lack of acetyl-coa in Hif-p4h-2 gt/gt tissues, which is presumably due to pyruvate dehydrogenase inhibition, is likely to contribute to the decreased lipogenesis (Supplementary Fig. 7). Extensive liver-specific stabilization of Hif-2a leads to hepatic steatosis (26,28). However, the Hif-p4h-2 gt/gt mice in the present study showed no steatosis but were instead protected against it. Liver-specific stabilization of Hif-2a by acute Hif-p4h-3 deletion likewise did not lead to hepatic steatosis, suggesting that low-level hepatic Hif-2a stabilization, as found in the present Hif-p4h-2 gt/gt mice, has beneficial effects, whereas extensive hepatic Hif-2a stabilization leads to steatosis (26,28). Liver-specific stabilization of Hif-1a and Hif-2a appears to have no effect on hepatic cholesterol synthesis or intestinal cholesterol absorption, but extensive liverspecific Hif-2a stabilization increases hepatic and serum cholesterol levels (28,29) as a result of decreased cholesterol oxidation to bile acids (29). However, the Hif-p4h- 2 gt/gt mice with low-level hepatic stabilization of Hif-2a had decreased serum cholesterol levels. The decreased amount of acetyl-coa is likely to contribute to the low serum cholesterol level in the Hif-p4h-2 gt/gt mice (Supplementary Fig. 7), but other mechanisms may also be involved. Mice with adipocyte-specific Hif-p4h-2 deletion also have less WAT, smaller adipocytes, a lower number of adipose tissue macrophages, and improved glucose tolerance (25); however, such changes were seen only in those fed an HFD, and no changes were reported in serum cholesterol levels, suggesting that Hif-p4h-2 deficiencies in several tissues play an important role in metabolic changes in Hif-p4h-2 gt/gt mice. Acute hepatic Hif-p4h-3 deletion has also been reported to improve glucose tolerance and insulin sensitivity, but no data were available on its effects on weight gain or serum cholesterol levels (26). Inhibition of Hif-1a by disruption of its gene in adipocytes (30) or administration of its inhibitor (31) or antisense oligonucleotides (32) attenuates the consequences of an HFD in mice. Currently, no explanation is available for the discrepancy between those data and the beneficial effects of Hif-a stabilization by Hif-p4h-2 deficiency, but the additional stabilization of Hif-2a has been suggested to possibly play an important role (25). Administration of FG-4497 to mice in two models of metabolic dysfunction led to changes very similar to those seen in the Hif-p4h-2 gt/gt mice, indicating that HIF-P4H-2 inhibition may not only protect against the development of obesity and metabolic dysfunction but also reverse them. FG-4497 inhibits all three HIF-P4Hs, but in view of the changes found in the Hif-p4h-2 gt/gt mice, it would seem possible to obtain a similar effect with a compound that specifically inhibits HIF-P4H-2. Of note, administration of another pan-hif-p4h-inhibitor, FG-4592, currently in clinical trials for treatment of anemia of chronic kidney disease, also lowers serum cholesterol levels and increases the HDL/LDL ratio (33,34), thus supporting the view that HIF-P4H-2 inhibition may indeed

10 diabetes.diabetesjournals.org Rahtu-Korpela and Associates 3333 be a useful strategy for the treatment of obesity and its consequences. Acknowledgments. The authors thank T. Aatsinki, R. Juntunen, E. Lehtimäki, S. Rannikko, and M. Siurua for excellent technical assistance. Funding. This study was supported by Academy of Finland grants and (to J.M.); Center of Excellence grant (to J.M.); the S. Jusélius Foundation (to J.M. and P.K.); Academy of Finland grants , , , and (to P.K.); and the Emil Aaltonen Foundation (to P.K.). Duality of Interest. G.W. is a senior cell biology director at FibroGen, Inc. K.I.K. is a scientific founder and consultant of FibroGen, Inc., which develops HIF- P4H inhibitors as potential therapeutics. K.I.K. and J.M. own equity in this company, and the company has sponsored research in the laboratory headed by K.I.K. and currently supports research headed by J.M. No other potential conflicts of interest relevant to this article were reported. Author Contributions. L.R.-K., S.K., and R.S. contributed to the research and data analysis. S.H. contributed expertise in serum lipid analyses. R.B.S. and E.L. contributed to the MRI analyses. K.A.M. and K.-H.H. contributed to the metabolic home cage experiments and analysis. G.W. provided the FG-4497 and made useful suggestions. K.I.K. contributed to generating the Hif-p4h-2 gt/gt mouse line and to the study design, data analysis, and writing of the manuscript. J.M. contributed to generating the Hif-p4h-2 gt/gt mouse line and to the discussions. P.K. contributed to generating the Hif-p4h-2 gt/gt mouse line and study supervision and to the study design, data analysis, and writing of the manuscript. P.K. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. References 1. Kaelin WG Jr, Ratcliffe PJ. 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