Journal Club

1. Journal Club 亀田メディカルセンター　糖尿病内分泌内科 Diabetes and Endocrine Department, Kameda Medical Center 松田　昌文 Matsuda, Masafumi 2007年12月13日　8:20-8:50 B棟８階　カンファレンス室

2. 1Sirtris Pharmaceuticals Inc., 790 Memorial Drive, Cambridge, Massachusetts 02139, USA. 2 Division of Endocrinology and Metabolism, Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA. 3 Department of Pathology, Paul F. Glenn Laboratories for the Biological Mechanisms of Aging, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.

3. Sirt1: a metabolic master switch that modulates lifespan By regulating transcriptional co-regulators such as PGC-1aor by directly interacting with transcription factors, Sirt1 can modulate gene-expression profiles in target tissues such as brain, liver, fat, pancreatic beta cells and muscle. By decoding NAD+ fluctuations in these tissues, Sirt1 might link the nutritional status of the cell to the regulation of its metabolism. Whereas the impact of Sirt1 in tissues such as liver (increased glucose output via gluconeogenesis), fat (lipolysis and mobilization of free fatty acid) and pancreatic beta cells (increased glucose-stimulated release of insulin) has been shown, its effect on metabolism in brain and skeletal muscle is still elusive. Nature Medicine 12, 34 - 36 (2006)

4. Background and Aim Calorie restriction extends lifespan and produces a metabolic profile desirable for treating diseases of ageing such as type 2 diabetes. SIRT1, an NAD1-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity. Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival. Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol.

5. Methods The compounds, SRT1460, SRT1720, and SRT2183, were synthesized at Sirtris Pharmaceuticals. SIRT1 constructs were expressed in E. coli and purified by affinity, size exclusion, and/or ion exchange chromatography. The SIRT1 fluorescence polarization assay used a peptide based on p53 linked to biotin at the N-terminal end and to a fluorescent tag at the C-terminal end. The reaction was a coupled enzyme assay where the first reaction was the deacetylation reaction and the second was cleavage by trypsin at the exposed lysine. The mass spectrometry assay was used for all SAR studies and employs the same peptide as the HTS assay. For the in cell western assay (ICW) the acetylation state of p53 was determined using a polyclonal antibody against the acetylated lysine residue at position 382 in p53. The effect of test compounds on the Km of SIRT1 for peptide substrate was examined using the mass spectrometry assay. Isothermal titration calorimetry was performed using a VP-ITC (MicroCal, Inc.). For the isobologram analysis a concentration matrix of two compounds was created and tested against SIRT1 using the mass spectrometry assay. For pharmacokinetic studies compounds were administered by oral gavage or intravenously to C57BL/6 male mice at the doses indicated. For all in vivo efficacy studies vehicle or test compound was administered once daily by oral gavage. For the DIO studies, C57BL/6 male mice were fed a high-fat diet until their mean body weight reached approximately 40 g. Lepob/ob mice and heterozygous Lepob1 mice were placed on a high-fat diet for a minimum of 1 week before the start of a study and remained on the high-fat diet for the duration of the study. Male fatty (fa/fa) Zucker (ZF) rats (Harlan Sprague–Dawley, Inc.) were used for the studies.

6. Figure 1 | Identification of potent SIRT1 activators unrelated toresveratrol. a, Chemical structures of SIRT1 activators, resveratrol, SRT1460, SRT2183, and SRT1720.

7. Figure 1 | Identification of potent SIRT1 activators unrelated toresveratrol. b, The effect of activators on human SIRT1 enzyme activity measured by mass spectrometry. c, Cellular activity was measured using an ICW that monitors the degree of p53 deacetylation in U2OS cells using b-tubulin as a normalization control. Compounds were tested at concentrations of: resveratrol (100 mM), SRT2183 (10 mM), SRT1460 (10 mM), SRT1720 (0.10 mM). Each concentration represents the approximate EC50 for each compound. n=6 for all compounds tested except for resveratrol, where n=3. Data are expressed as mean±s.d. The activation of SIRT1 resulting in p53 deacetylation could be blocked by a SIRT1 inhibitor, 6-chloro-2,3,4,9-tetrahydro-1-H-carbazole-1-carboxamide (10 mM). n=3 for all compounds tested.

8. Figure 2 | In vitro characterization ofactivators of human SIRT1. a, The effect of SIRT1 activators on peptide substrate Km. b, Calorimetric titrations of SIRT1-C–peptide substrate complex with the activator SRT1460. Top panel: heat of binding SRT1460 to enzyme–peptide complex. Bottom panel: integrated fit with a one-site binding model.

9. Figure 2 | In vitro characterization of activators of human SIRT1. c, Isobologram analysis of resveratrol versus SRT1720 and SRT1720 versus SRT1460. The experimental data are best fit to the theoretical line of additivity (dashed line). d, SIRT1 N-terminal truncations define the allosteric compound binding site. The ability of resveratrol and SRT1720 to activate SIRT1 was examined against a series of N-terminal deletions in the mass spectrometry assay.

10. Figure 3 | SIRT1 activators in mouse models of type 2 diabetes. a, Plasma levels of SRT1720 after administration by oral gavage. b, Effect of SRT1720 treatment over 10 weeks on fed plasma glucose levels in DIO mice.

11. Figure 3 | SIRT1 activators in mouse models of type 2 diabetes. c, Glucose excursion during an intraperitoneal glucose tolerance test (5 weeks) in DIO mice treated with the indicated compounds. d, Plasma insulin levels after 10 weeks treatment with indicated compounds.

12. Figure 3 | SIRT1 activators in mouse models of type 2 diabetes. e, Glucose response in DIOmice during an insulin tolerance test after 10 weeks treatment with the indicated compounds. f, Skeletal muscle citrate synthase activity (Vmax mg21 protein, 11 weeks, n=55). All studies consisted of ten mice per group unless noted. Statistics were conducted as an ANOVA; asterisk P<0.05, double asterisk P<0.01 and triple asterisk P<0.001. Data are expressed as mean±s.e.m.

13. Figure 3 | SIRT1 activators in mouse models of type 2 diabetes. g, Effect of SRT1720 treatment in diabetic Lepob/ob mice (1 week). h, Effect of SRT501 treatment (1,000 mg per kg (body weight)) in diabetic Lep ob/ob mice after 2 weeks. i, Effect of SRT501 treatment (500 mg per kg (body weight), 4 weeks) in DIO mice.

14. Figure 4 | SIRT1 activator SRT1720 in the Zucker fa/fa rat model. a, Postabsorptive blood glucose (3 weeks). b, c Glucose and insulin responses during an oral glucose tolerance test (3.5 weeks, ANOVA). After 4 weeks a hyperinsulinaemic-euglycaemic clamp study was conducted.

15. Figure 4 | SIRT1 activator SRT1720 in the Zucker fa/fa rat model. (d–f) Glucose infusion rate (GIR), glucose disposal rate (GDR) and insulin-stimulated glucose disposal rate (IS-GDR) were significantly enhanced. g, Hepatic glucose output (HGO) suppression (%).

16. Figure 4 | SIRT1 activator SRT1720 in the Zucker fa/fa rat model. h, Plasma fatty acid concentration during the clamp was significantly lower in SRT1720-treated animals (ANOVA). B, basal conditions. C, clamp conditions. i, Glucose excursion during a PTT (pyruvate tolerance test) was reduced, indicating reduced gluconeogenic capacity.

17. Summary and Conclusion In diet-induced obese and geneticallyobese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes.

19. Howard Hughes Medical Institute, Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037,

20. Background Osteoclasts are bone-resorbing cells derived from hematopoietic precursors of the monocyte-macrophage lineage. Regulation of osteoclast function is central to the understanding of bone diseases such as osteoporosis, rheumatoid arthritis and osteopetrosis. Although peroxisome proliferator–activated receptor-g (PPAR-g) has been shown to inhibit osteoblast differentiation, its role, if any, in osteoclasts is unknown. This is a clinically crucial question because PPAR-g agonists, ‘‘such as thiazolidinediones—’’ a class of insulin-sensitizing drugs, have been reported to cause a higher rate of fractures in human patients.

21. Figure 1 Tie2Cre-mediated PPAR-g deletion leads to extramedullary hematopoiesis in the spleen. • Genotype PCR analysis of various tissues of flox/null mice with Tie2Cre (f/–Tie2Cre+/) or without Tie2Cre (f/–). The null allele was a recombined floxed allele and served as an internal DNA loading control. Tie2Cre converted the other floxed allele into a null allele in all hematopoietic tissues (yellow boxes) without affecting mesenchymal tissues. WAT, white adipose tissue; LN, lymph node; BAT, brown adipose tissue; BM, bone marrow. • Gross morphology (left) and histology (right) of the spleen. Arrows indicate megakarocytes; scale bar represents 50 mm. • Pale bones in the mutants (+Cre).

22. 10–18-week-old adults 18-d 5-d (d) Spleen–to–body weight ratio in 10–18-week-old adults (left, n=13) and in 18-d-old (middle, n =6) and 5-d-old (right, n=6) pups. (e) Total cell number (top, n=9), progenitor cell number (middle, n=14) and hematopoietic stem cell percentage (bottom, n=8) in bone marrow (left) and spleen (right). BM, bone marrow; E, erythroid; GM, granulocyte/macrophage. Figure 1 Tie2Cre-mediated PPAR-g deletion leads to extramedullary hematopoiesis in the spleen.

23. (f) Expression of transcription factors regulating early hematopoiesis in bone marrow and spleen. Expression was normalized to that of b-actin. Tal1, T-cell acute lymphocytic leukemia 1; Lmo2, LIM domain only 2; Fog1, Friend of GATA-1; Gata1, GATA binding protein 1; Gata2, GATA binding protein 2. (g) Complete blood cell count. RBC, red blood cell; HGB, hemoglobin; PL, platelet. (h) White blood cell differential count. All changes in blood cell counts were not significant (n=9, P < 0.05). NE, neutrophil; LY, lymphocyte; MO, monocyte; EO, eosinophil; BA, basophil. *P < 0.05; ***P < 0.005. Figure 1 Tie2Cre-mediated PPAR-g deletion leads to extramedullary hematopoiesis in the spleen.

24. BONE! Bone Volume Fraction Bone Mineral Density (a) mCT analysis of the femur and tibia from 3-month-old wild-type (–Cre) and mutant (+Cre) mice. BVF (top) and BMD (bottom) were increased for both the proximal (Prox) and the distal (Dist) regions in the mutants (n = 3). Figure 2 Tie2Cre-mediated PPAR-g deletion leads to osteopetrosis represented by increased bone volume, decreased medullary cavity space and reduced bone resorption.

25. (b) Static histomorphometric analyses of the femur from 2-monthold mice. BV / TV, trabecular bone volume / total tissue volume; Tb.th, trabecular thickness; Tb.n, trabecular number; Tb.sp, trabecular separation (n = 4; error bars, s.e.m.). Figure 2 Tie2Cre-mediated PPAR-g deletion leads to osteopetrosis represented by increased bone volume, decreased medullary cavity space and reduced bone resorption.

26. (c) mCT images of the proximal region of femurs (top), whole-body X-ray analyses with enlarged images for femurs and knee joints (middle, arrow indicates the decreased size of the medullary cavity in the +Cre femur) and H & E stained femur sections imaged under transmitted polarized light (bottom). osteopetrosis osteopetrosis Figure 2 Tie2Cre-mediated PPAR-g deletion leads to osteopetrosis represented by increased bone volume, decreased medullary cavity space and reduced bone resorption.

27. Greek klastas “broken (d) TRAP staining of osteoclasts (arrows) in femur sections. PPAR-g inhibit osteoblast differentiation Figure 2 Tie2Cre-mediated PPAR-g deletion leads to osteopetrosis represented by increased bone volume, decreased medullary cavity space and reduced bone resorption.

28. (e) Alkaline phosphatase staining of osteoblasts (arrows) in femur sections. B, bone. Bone formation rate unchanged Figure 2 Tie2Cre-mediated PPAR-g deletion leads to osteopetrosis represented by increased bone volume, decreased medullary cavity space and reduced bone resorption.

29. Greek klastas “broken (f) Quantification of osteoclast and osteoblast cells. N.Oc/mm2, number of osteoclasts per tissue area; Ob.S/BS%, osteoblast surface per bone surface; N.Ob/B.Pm, number of osteoblasts per bone perimeter (mm–1). (g) Dynamic histomorphometry shows bone formation rate (BFR / BS) and mineral apposition rate (MAR) in 2-month-old mice, as measured by calcein double labeling (n = 4; error bars, s.e.m.). (h) Detection of bone resorption and bone formation markers. Urine deoxypyridinoline (DPD) bone resorption marker (left, n = 20) and serum alkaline phosphatase (middle) and osteocalcin (right) bone formation markers (n = 17) were quantified and plotted as shown. Error bars, s.e.m. *P < 0.05; **P < 0.01; ***P < 0.005.

30. ROSIGLITAZONE Receptor activagtor of nuclear facgtor-kB ligand • In vitro RANKL-mediated osteoclast differentiation from splenocytes. Mature osteoclasts were identified as multinucleated TRAP+ cells; • scale bar represents 50 mm. Figure 3 PPAR-g and ligand promote osteoclast differentiation by regulating c-fos expression.

31. (b) mRNA expression of osteoclast-specific functional genes (left), RANKL-induced transcription factors (middle) and mature macrophage-related inflammatory genes (right). (c) mRNA expression of osteoclast genes were suppressed by PPAR-g antagonists GW9662 and T0070907 only in WT cells. Figure 3 PPAR-g and ligand promote osteoclast differentiation by regulating c-fos expression.

32. (d) mRNA expression during a time course after RANKL treatment. The results are shown as relative mRNA levels to the ribosomal protein L19. BRL or antagonists were used at 1 mM. Acp5, TRAP; Calcr, calcitonin receptor; Car2, carbonic anhydrase 2; Ctsk, cathepsin K; Mmp9, matrix metallopeptidase-9; Fos, c-fos osteosarcoma oncogene; Fosl1, fos-like antigen 1; Nfatc1, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1; Ccl2, monocyte chemoattractant protein-1; Tnfa, tumor necrosis factor-a. Figure 3 PPAR-g and ligand promote osteoclast differentiation by regulating c-fos expression.

33. (e) RANKL-induced c-Jun hosphorylation or IkBa degradation was not decreased in the mutant cells, as detected by western blotting. Figure 3 PPAR-g and ligand promote osteoclast differentiation by regulating c-fos expression.

34. (f) Identification of PPREs in the c-fos promoter. The c-fos promoter was induced by PPAR-g and ligand in transient transfection assays in RAW264.7 cells, as determined by luciferase reporter assays (left). Luciferase reporter assays with truncated c-fos promoters indicated the presence of two PPREs (middle). The two numbers for fold induction represent (g+a/BRL) / (CMX/veh) followed by (g+a/BRL) / (g+a/veh). Right, alignment of mouse (m), rat (r) and human (h) c-fosA and c-fosB PPRE regions, together with known PPREs in ap2 (adipocyte-specific fatty-acid binding protein), PEPCK (phosphoenolpyruvate carboxykinase), ACS (acyl-CoA synthase) and DR-1 (direct repeat-1) consensus sequence. b-gal, b-galactosidase. Figure 3 PPAR-g and ligand promote osteoclast differentiation by regulating c-fos expression.

35. (g) ChIP analysis of PPAR-g binding to the c-fosA, c-fosB or upstream promoter region (c-fos –5 Kb, as negative control) in bone marrow–differentiated monocytes and macrophages. E8 and H100 denote the antibodies to PPAR-g described in the Methods. Figure 3 PPAR-g and ligand promote osteoclast differentiation by regulating c-fos expression.

36. (h) A schematic diagram of RANKL signaling pathways, showing PPAR-g and its ligand promoting osteoclast differentiation by specifically regulating the c-fos pathway. Figure 3 PPAR-g and ligand promote osteoclast differentiation by regulating c-fos expression.

37. CONTROL 5x (a,b) Osteoclast differentiation blockade can be rescued by ectopic expression of c-fos by retroviral gene transfer. (a) Number of multinucleated osteoclasts as a percentage of the number of GFP+ cells in mutant cells infected with c-fos virus (MIGR-cfos) or control virus (MIGR-F); scale bar represents 50 mm. (b) Ectopic expression of c-fos rescues the RANKL-mediated, but not BRL-stimulated, induction of osteoclast genes in the mutant cells. The results are shown as relative mRNA levels to the ribosomal protein L19. Tnfrsf11a, Tumor necrosis factor receptor superfamily, member 11a, or RANK. Figure 4 In vitro and in vivo rescues of osteoclast differentiation and extramedullary hematopoiesis.

38. osteoclast differentiation and extramedullary hematopoiesis (c,d) Extramedullary hematopoiesis can be reversed by bone marrow transplantation. (c) Extramedullary hematopoiesis can be conferred by transplantation of mutant (+Cre) bone marrow into WT recipients (–Cre; n = 5). (d) Extramedullary hematopoiesis can be rescued by transplantation of WT (–Cre) bone marrow into mutant recipients (+Cre; n = 8). Extramedullary hematopoiesis was determined by spleen–to–body weight ratio, spleen cell number and bone marrow cell number. Transplantation efficiency was measured by FACS analysis of GFP+ population in blood cells. *P < 0.05; ***P < 0.005. Figure 4 In vitro and in vivo rescues of osteoclast differentiation and extramedullary hematopoiesis.

39. Summary This study reveals an unexpected role for PPAR-g and its ligand in promoting osteoclast differentiation and bone resorption. Loss of function by targeted PPAR-g deletion impairs osteoclast differentiation and bone resorption, resulting in osteopetrosis and extramedullary hematopoiesis. In contrast, gain of function by ligand activation of PPAR-g accelerates osteoclast differentiation and bone resorption in a receptor-dependent manner.

40. Conclusion These findings have potential clinical implications, as they suggest that long-term rosiglitazone usage in the treatment of type 2 diabetes and insulin resistance may cause osteoporosis, owing to a combination of decreased bone formation and increased bone resorption. They also suggest that selective PPAR-g modulators may provide a new strategy for the treatment of bone diseases associated with increased osteoclast activity, such as osteoporosis and rheumatoid arthritis.