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Journal Club

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７月 12 日　 8:20-8:50 B 棟８階　カンファレンス室. AⅡ. PI 3-kinase. AT-R. P85. P-Ser-. P110. Insulin Receptor. P-Ser-. -Tyr-P. -Tyr-P. β. α. -Tyr-P. -Tyr-P. -Tyr-P.

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Journal Club

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  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７月12日　8:20-8:50 B棟８階　カンファレンス室

  2. AⅡ PI 3-kinase AT-R P85 P-Ser- P110 Insulin Receptor P-Ser- -Tyr-P -Tyr-P β α -Tyr-P -Tyr-P -Tyr-P P-Ser- IRS-1 Diagram of AⅡsignaling interactions with the insulin receptor, IRS-1, and PI 3 kinase in RASMC Folli F et al: J Clin Invest 100:2158, 1997

  3. Chemical Structure of ARBs Tetrazole structure Non-tetrazole structure

  4. Telmisartan vs Pioglitazone Telmisartan Pioglitazone Binding mode of telmisartan in the PPARLBD. Hypertension. 2004;43:1-10.

  5. Effect of PPARg Improvement of Insulin Sensitivity PPAR Ligand Pioglitazone Liver (Fatty Liver Improvement) Adiponectin↑ b TNF-a↓ Reduced Oxidative Stress Pancreas Muscle (Less TG) Adipocytes (Anti-adiposopathy) Beta cell function Preservation Improvement Glucotoxicity Lipotoxicity

  6. Effect of Non-tetrazole ARB (Telmisartan) Improvement of Insulin Sensitivity AII Antagonist Telmisartan Liver (Fatty Liver Improvement) Adiponectin↑ b TNF-a↓ Reduced Oxidative Stress Pancreas Muscle (Less TG) Adipocytes (Anti-adiposopathy) Beta cell function Preservation Improvement Glucotoxicity Lipotoxicity

  7. AIM To evaluate the efficacy of an ARB in preventing transition from microalbuminuria to overt nephropathy in Japanese patients

  8. Criteria for subjects Inclusion criteria: • Age: from 30 to 74 years • Type 2 diabetes • Urinary albumin-to-creatinine ratio (UACR) 100–300 mg/g • Serum creatinine <1.5 mg/dl (men) and <1.3 mg/dl (women) Exclusion criteria: • type 1 diabetes, • age of diabetes onset <30 years, • seated systolic blood pressure (SBP)/diastolic blood pressure (DBP) ≧180/100 mmHg • definable chronic kidney disease other than diabetic nephropathy.

  9. PROTCOL • A total of 527 patients out of 1,855 screened were randomized. • Groups: 80 or 40 mg telmisartan or placebo • The starting dose was 20 mg, titrated to 40 mg after 2 weeks or to 80 mg after a further 2 weeks. • Minimum treatment period was 1 year for each patient.

  10. SUBJECTS Of the 527 randomized patients (mean age 61.7 years), 13 were excluded from primary analysis because of suspected type 1 diabetes or UACR measurements being missing during treatment. Mean duration of follow-up was 1.3 ± 0.5 years (maximum 2.3 years).

  11. Kaplan-Meier curves for transition from incipient to overt nephropathy in patients treated once daily with 80 mg telmisartan (T80), 40 mg telmisartan (T40), and placebo. Mean duration of follow-up was 1.3 0.5 years (maximum 2.3 years) n=514 Transition rates to overt nephropathy were 80 mg telmisartan (n = 168) 16.7%, 40 mg telmisartan (n = 172) 22.6%, and placebo (n = 174) 49.9% (both telmisartan doses vs. placebo, P <0.0001)

  12. Kaplan-Meier curves for transition from incipient to overt nephropathy in patients treated once daily with 80 mg telmisartan (T80), 40 mg telmisartan (T40), and placebo. 163 normotensive patients n=163 Transition rates in normotensive patients were 80 mg telmisartan (n = 51) 11.0%, 40 mg telmisartan (n = 58) 21.0%, and placebo (n = 54) 44.2% (both telmisartan doses vs. placebo, P < 0.01)

  13. One or more adverse event was recorded in 90% of patients in each treatment group; most events were mild or moderate in intensity.

  14. Summary and Conclusion • In Japanese type 2 diabetic patients, achievement of microalbuminuria remission was superior with 80 or 40 mg telmisartan than with placebo. • Telmisartan reduced transition from incipient to overt nephropathy. • Telmisartan also reduced transition to overt nephropathy in normotensive patients, suggesting telmisartan had a blood pressure–independent effect.

  15. 1Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, Massachusetts 02115, USA. 2Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA. 3Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA. 4Present address: Department of Medicine, Division of Endocrinology, Metabolism, and Nutrition, Duke University Medical Center, Durham, North Carolina 27704, USA. †Present address: Clinical Molecular Biology, Teikyo University, Kanagawa 199-0195, Japan

  16. Background Lipid chaperones (also known as fatty acid binding proteins) are important links between metabolic and inflammatory responses. In particular, genetic loss of function of the fatty acid binding protein aP2 is known to protect mice against many components of metabolic syndrome.

  17. Model of FABPs at the intersection of inflammatory and metabolic signal cascades

  18. Hypothesis Pharmacological agents that modify FABP function may offer therapeutic opportunities for many components of metabolic syndrome, such as insulin resistance, type 2 diabetes, and atherosclerosis.

  19. BMS309403 is a rationally designed, potent, and selective inhibitor of aP2 that interacts with the fatty-acid binding pocket within the interior of the protein and competitively inhibits the binding of endogenous fatty acids.

  20. FABP4 FABP5 Figure 1 | Target-specific effects of aP2 inhibition on MCP-1 production in macrophages. b, Protein levels of aP2 and mal1 in human THP-1 [a human monocytic leukaemia cell line] macrophages and mouse macrophage cell lines, aP2+/+, aP-/- and aP2-/-R. c, aP2 and mal1 mRNA levels analysed by quantitative real-time PCR. d, MCP-1 [monocyte chemoattractant protein] production in human THP-1 macrophages treated with aP2 inhibitor at the indicated concentrations for 24 h. e, MCP-1 production in mouse cell lines treated with the aP2 inhibitor at the indicated concentrations for 24 h. Data are shown as the mean±s.e.m. *P<0.05, **P<0.01 compared with the control (each untreated cell line). AU, arbitrary units.

  21. Figure S2: Atherosclerosis in Apoe-/- mice treated with the aP2 inhibitor. a, Experimental design of the early intervention study and en face aortas stained with Sudan IV. b, Quantitative analyses of the atherosclerotic lesion areas (% of total aorta surface area) in the en face aorta were performed in both vehicle (n= 12) and aP2 inhibitor (n = 13) groups. c, Atherosclerotic lesions in the aortic root at the level of aortic valves were stained with Oil Red O. Magnification, x40. d,immunohistochemical detection of macrophages (MOMA-2) in the aortic root at the level of aortic valves was determined. Magnification, x40. e, Quantitative analysis of the atherosclerotic lesion areas in the proximal aorta were performed in both the vehicle (n= 7) and aP2 inhibitor (n = 9) groups. f, Lipoprotein profile in Apoe-/- mice treated with the vehicle (red circle) and aP2 inhibitor (blue circle) in the early intervention study. Data are presented as an average (n = 3) percent distribution of total cholesterol for each group. Data are expressed as the mean ± S.E. *P < 0.05, **P < 0.01. VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein.

  22. Figure 2 | Atherosclerosis in Apoe-/- mice treated with the aP2 inhibitor. a, Experimental design of the late intervention study and en face aortas stained with Sudan IV. b, Quantitative analyses of the atherosclerotic lesion areas (per cent of total aorta surface area) in the vehicle (n=16) and aP2 inhibitor (n=15) groups. c, d, Oil Red O (c) and MOMA-2 (d) stainings of atherosclerotic lesions in the aortic root at the level of the aortic valves. Magnification, x40. e, Quantitative analyses of the proximal aorta atherosclerotic lesion areas in the vehicle (n=11) and aP2 inhibitor (n=6) groups. f, Lipoprotein profile in Apoe-/- mice treated with vehicle (red) and aP2 inhibitor (blue) in the late intervention study. Data are presented as an average (n=3) per cent distribution of total cholesterol for each group. Data are expressed as the mean ± s.e.m. *P<0.01.

  23. Figure 3 | Effects of aP2 inhibitor on lipid accumulation, in macrophages. a, Oil Red O staining of THP-1 macrophage foam cells loaded with acetylated low density lipoprotein (50 mgml-1) in the absence or presence of aP2 inhibitor (25 mM). Magnification, x400. b, c, Cholesterol ester levels normalized to cellular protein content in human THP-1 macrophages (b) and mouse macrophage cell lines, aP2+/+, aP2-/- and aP2-/-R (c).

  24. d, e, APOA1-specific cholesterol efflux in THP-1 macrophages (d) and mouse cell lines (e) in the absence or presence of aP2 inhibitor (25 mM). Using 3H-cholesterol

  25. f–j, Expression of Acat1 (f) and chemoattractant and inflammatory cytokines, Mcp-1 (g), Il1b (h), Il6 (i), and Tnf (j) in macrophages normalized to 18s rRNA levels. Data are normalized to untreated cells and expressed as the mean±s.e.m. *P<0.05, **P<0.01 compared with the control (each untreated cell line). DMSO, dimethyl sulphoxide.

  26. Figure 4 | Metabolic studies in aP2-inhibitor-treated adipocytes a, Oil Red O staining of wild-type (WT), FABP-deficient (KO), FABPdeficient reconstituted with aP2 (KO+aP2), and FABP-deficient with vector (KO+GFP) adipocyte cell lines. b, Fatty acid uptake using 3Hstearate in adipocyte cell lines.

  27. 40mg/kg per day for 6 weeks Figure 4 | Metabolic studies in aP2-inhibitor-treated ob/ob mice. c, Blood glucose levels in ob/ob mice treated with vehicle (n=6) or aP2 inhibitor (n=6) at the fed state after 2 weeks of treatment and at the fasting state after 6 weeks of treatment. d, e, Plasma levels of insulin (d) and adiponectin (e) in ob/ob mice treated with vehicle (n=6) or aP2 inhibitor (n=6) for 6 weeks Data are shown as the mean±s.e.m. *P<0.05, **P<0.01.

  28. 0.5g/kg 2U /kg f, Glucose tolerance tests performed after 4 weeks of treatment in ob/ob mice with vehicle (open circle, n=6) or aP2 inhibitor (closed circle, n=6). g, Insulin tolerance tests performed after 5 weeks of treatment in ob/ob mice with vehicle (open circle, n=6) or aP2 inhibitor (closed circle, n=6). Data are shown as the mean±s.e.m. *P<0.05, **P<0.01.

  29. (75min) (120min) h–j, Hyperinsulinaemic[12.5mU/kg per min]–euglycaemic clamp studies performed in ob/ob mice treated with vehicle (n=7) or aP2 inhibitor (n=9) for 4 weeks. Basal and clamp hepatic glucose production (HGP) (h), glucose disposal rate (Rd) and glucose infusion rate (GIR) (i), and tissue glucose uptake in gastrocnemius muscle and epididymal fat (j). Data are shown as the mean±s.e.m. *P<0.05, **P<0.01.

  30. Figure 5 | Effects of aP2 inhibitor in adipose tissue of ob/ob mice. a, Haematoxylin and eosin staining of the adipose tissue in ob/ob mice treated with vehicle or aP2 inhibitor. Scale bar, 200 mm.

  31. Macrophage markers b–g, Expression of F4/80 (b), Cd68 (c), Mcp-1 (d), Il1b (e), Il6 (f), and Tnf (g) in the adipose tissue of ob/ob mice treated with vehicle (n=6) or aP2 inhibitor (n=6).

  32. h, JNK1 activity in the adipose tissue of ob/ob mice. Quantification is shown in the graph below. i, Insulin-stimulated IRb tyrosine 1162/1163 and AKT serine 473 phosphorylation (p) in the adipose tissues of ob/ob mice. The graphs on the right of each blot show the quantification. Data are shown as the mean±s.e.m. *P<0.05, **P<0.01.

  33. DAIICHI PHARM Cell Signaling

  34. Figure 6 | Effects of aP2 inhibitor in liver of ob/ob mice. b, Haematoxylin and eosin staining of the liver of ob/ob mice treated with vehicle or aP2 inhibitor. Scale bar, 200 mm.

  35. Stearoyl-CoA desaturase 1, Fatty acid synthase, Acetyl CoA carbodylase 1 Figure 6 | Effects of aP2 inhibitor in liver of ob/ob mice. a, aP2 and mal1 protein in the adipose tissue of ob/ob mice treated with vehicle or aP2 inhibitor. For control, the adipose tissue of ob/ob;aP2+/+ (A+) and ob/ob; aP2-/- (A-) mice was used. c–f, Triglyceride (TG) content (c) and mRNA expression of Scd1 (d), Fasn (e), and Acaca (f) in the liver of ob/ob mice treated with vehicle (n=6) or aP2 inhibitor (n=6).

  36. g, JNK1 activity in the liver of ob/ob mice treated with vehicle or aP2 inhibitor. The graph below the blot shows quantification. h, Insulin-stimulated IRb tyrosine 1162/1163 and AKT serine 473 phosphorylation in the liver tissues of ob/ob mice treated with vehicle or aP2 inhibitor. The graphs demonstrate the quantification of phosphorylation of each molecule. Data are shown as the mean±s.e.m. *P<0.05.

  37. Summary and Conclusion We have provided a critical proof of principle in mice that aP2 could be successfully targeted by an orally active, small molecule inhibitor to generate a profile reminiscent of genetic deficiency in vitro and in vivo. The metabolic function of aP2 in humans may be similar to that observed in mouse models. It is therefore possible that chemical inhibition of aP2 in humans might also show beneficial effects against diabetes and cardiovascular disease.

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