Journal Club

1. Journal Club GillumMP, Kotas ME, Erion DM, Kursawe R, Chatterjee P, Nead KT, Muise ES, Hsiao JJ, Frederick DW, Yonemitsu S, Banks AS, Qiang L, Bhanot S, Olefsky JM, Sears DD, Caprio S, Shulman GI. SirT1 Regulates Adipose Tissue Inflammation. Diabetes. 2011 Dec;60(12):3235-45. • 埼玉医科大学　総合医療センター　内分泌・糖尿病内科 • Department of Endocrinology and Diabetes, • Saitama Medical Center, Saitama Medical University • 松田　昌文 • Matsuda, Masafumi 2011年12月15日8:30-8:55 ８階　医局

2. sirtuin (silent mating type information regulation 2 homolog) 1 NAD-dependent deacetylase sirtuin-1 Sirtuin 1 is downregulated in cells that have high insulin resistance and inducing its expression increases insulin sensitivity, suggesting the molecule is associated with improving insulin sensitivity. Activators Resveratrolhas been claimed to be an activator of Sirtuin1,however this has been disputed. Studies show that resveratrol increases the expression level of SIRT1, meaning that it does increase the activity of SIRT1, though not necessarily by direct activation. SRT-1720 was also claimed to be an activator but this now has been questioned.

3. Homeostatic mechanisms in mammals respond to hormones and nutrients to maintain blood glucose levels within a narrow range. Caloric restriction causes many changes in glucose metabolism and extends lifespan; however, how this metabolism is connected to the ageing process is largely unknown. We show here that the Sir2 homologue, SIRT1—which modulates ageing in several species1–3—controls the gluconeogenic/glycolytic pathways in liver in response to fasting signals through the transcriptional coactivator PGC-1a. A nutrient signalling response that is mediated by pyruvate induces SIRT1 protein in liver during fasting. We find that once SIRT1 is induced, it interacts with and deacetylates PGC-1a at specific lysine residues in an NAD1- dependent manner. SIRT1 induces gluconeogenic genes and hepatic glucose output through PGC-1a, but does not regulate the effects of PGC-1aon mitochondrial genes. In addition, SIRT1 modulates the effects of PGC-1a repression of glycolytic genes in response to fasting and pyruvate. Thus, we have identified a molecular mechanism whereby SIRT1 functions in glucose homeostasis as a modulator of PGC-1a. These findings have strong implications for the basic pathways of energy homeostasis, diabetes and lifespan. Nature 434, 7029 (Mar 2005)

4. 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.

5. Sirt1 is a potential metabolic master switch. By regulating transcriptional co-regulators such as PGC-1α or 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 liver2 (increased glucose output via gluconeogenesis), fat7 (lipolysis and mobilization of free fatty acid) and pancreatic beta cells8 (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)

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

7. http://www.daiwa-grp.jp/dsh/results/33/pdf/08.pdf 線虫を飢餓. 状態にすると FOXO が活性化し、Dauer Formation という特殊な代謝状態を形成し生命を維持。細胞保護作用をもつタンパク質の転写が促進される 。 FOXO1を刺激し インスリン抵抗性を起す？！ 血糖上昇 他に アポトーシスを抑制 PPARγを抑制しインスリン抵抗性を起す 血糖上昇 飽食では逆 インスリン感受性がforkhead transcription factor： FOXOのレベルで調整される。これは、栄養・インスリンシグナルが低いときに活動させるnegativeな成長調整因子となる。逆にFOXO発現が臓器的に減少することで、各臓器は低栄養での成長抑制が減弱し、栄養学的可塑性が減少する。FOXO発現は臓器特異的な栄養可塑性・インスリン感受性にとって必要で、臓器がFOXO発現により自立的に応答することとなる。 FOXO1はPdx1転写は抑制　インスリン分泌は低下。

8. The 1Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut; the 2Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut; the 3Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut; the 4Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut; the 5Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut; 6Merck, Rahway, New Jersey; the 7Department of Cell Biology, Harvard School of Medicine, Boston, Massachusetts; the 8Department of Medicine, Columbia University, New York, New York; 9Isis Pharmaceuticals, Carlsbad, California; and the 10Department of Medicine, Division of Endocrinology and Metabolism, University of California San Diego, San Diego, California. Diabetes60:3235–3245, 2011

9. OBJECTIVE—Macrophage recruitment to adipose tissue is a reproducible feature of obesity. However, the events that result in chemokine production and macrophage recruitment to adipose tissue during states of energetic excess are not clear. Sirtuin 1 (SirT1) is an essential nutrient-sensing histone deacetylase, which is increased by caloric restriction and reduced by overfeeding. We discovered that SirT1 depletion causes anorexia by stimulating production of inflammatory factors in white adipose tissue and thus posit that decreases in SirT1 link overnutrition and adipose tissue inflammation.

10. RESEARCH DESIGN AND METHODS—We used antisense oligonucleotides to reduce SirT1 to levels similar to those seen during overnutrition and studied SirT1-overexpressing transgenic mice and fat-specific SirT1 knockout animals. Finally, we analyzed subcutaneous adipose tissue biopsies from two independent cohorts of human subjects.

11. Animals Male Sprague Dawley rats (Charles River) or C57BL/6 J mice (The Jackson Laboratory) were given ad libitum access to food and water under a standard 12-h light/dark cycle. MyD88/TRIF2/2, MyD88/IRF32/2, Caspase-12/2, and op/op mice were provided by RuslanMedzhitov (Yale University). SirtT1 fl/fl mice were crossed to aP2-Cre. SirT1 transgenic mice (SirBACO) (15) were fed a highfat diet (D12492, Research Diets) for 6 weeks. Rodents were fed regular chow (Harlan 2018S), high-fat chow (Harlan TD93075), high-fructose chow (Harlan TD89247), or a combination of high-fat/high-fructose chow as indicated. Antisense oligonucleotides (ASOs) (14) were dosed twice per week at 1.25 mg (for mice) or 37.5 mg/kg (for rats). The T2DM model is described in Erion et al. (14). All experiments were conducted in accordance with Yale University’s Institutional Animal Care and Use Committee policy.

12. Antibodies. The following antibodies were used: SirT1 (Upstate Biotechnology); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling); p65 (Genway). Cytokine measurements. Obesity array (Phoenix Pharmaceuticals) and 7-plex electrochemiluminescent assay (Meso Scale Discovery) were performed on pooled plasma (n = 7–8/group) per the manufacturers’ instructions. Cytokines were verified in duplicate using commercially available kits (TNF-a OptiEIA, BD Biosciences; rat IL-10, R&D Systems). Membranes spotted with antibodies against 34 soluble cytokines (RayBiotech) were incubated with plasma and then developed by chemiluminescence and quantified by densitometry using ImageJ. Quantitative RT-PCR. RNA was extracted using the QiagenRNeasy kit. Transcript abundance was assessed by real-time PCR on a 7500 Fast Real-Time PCR System (Applied Biosystems) and analyzed by DDCt method (primers available by request). NF-kB quantitative PCR arrays and reagents were purchased from SABiosciences. Fluorescence-activated cell sorting. Stromal vascular fractions from bilateral epididymal fat pads (5 mice/group) were digested as previously described (1) using LiberaseTM (Roche). Cells were stained with F4/80-APC and CD11b-PE (eBioscience), run on a FACSCalibur flow cytometer (BD Biosciences), and analyzed using FlowJo (Treestar).

13. Human studies. For adolescents, two gram samples of subcutaneous adipose tissue were obtained inferior to the umbilicus after local administration of 0.25% lidocaine with epinephrine, washed in PBS, and frozen in liquid nitrogen. Blood samples were obtained following an overnight fast (29). Homeostasis model assessment of insulin resistance (HOMA-IR) = [fasting glucose (mmol/L) ×fasting insulin (mU/L)]/405 (30). Whole-body composition was measured by dual-energy X-ray absorptiometry with a Hologic scanner. Magnetic resonance imaging studies were performed on a GE or Siemens Sonata 1.5 Tesla system (31,32). The nature and potential risks of the study were explained to all subjects before obtaining their written informed consent. The study was approved by the Yale University Human Investigation Committee. The adult subjects were studied as described in Sears et al. (33). Statistics. All values are expressed as the mean ±SEM. The significance between the mean values was evaluated by two-tailed unpaired Student t test or ANOVA.

14. FIG. 1. Knockdown of SirT1 mimicking that seen in obesity causes anorexia-driven weight loss and TNF-a elevation. A–C: Body weight (***P < 0.001) (n = 8–12/group) (A), anorexia (n = 12/group) (B), and loss of epididymal fat mass (n = 3–4/group) (C) of ad libitum fed rats treated with ASO biweekly for 1 month. D: Results from parallel measurement of plasma appetite regulatory hormones in animals treated with control or SirT1 ASO showing elevated TNF-a and possibly IL-6 in the SirT1 group (n = 7–8/group, pooled). HFD, high-fat diet.

15. FIG. 1. Knockdown of SirT1 mimicking that seen in obesity causes anorexia-driven weight loss and TNF-a elevation. E: Plasma TNF-a levels (n = 5/group). F: Adipose tissue levels of TNF-a (n = 3–4/group). G: SirT1 protein expression in WAT from animals treated with control ASO and fed normal chow, animals treated with SirT1 ASO and fed normal chow, and mildly diabetic animals fed high-fat/high-fructose chow. H: Quantification of representative data presented in F (n = 6/group). I: Plasma TNF-a levels in normal chow–fed control ASO–treated rats, normal chow–fed SirT1 ASO–treated rats, and highfat diet–fed, control ASO–treated rats by electrochemiluminescence (n = 4–5/group). HFD, high-fat diet.

16. FIG. 2. SirT1 knockdown/deletion causes adipose tissue inflammation and macrophage infiltration. A: Cytokine array performed on plasma from normal chow–fed control ASO– and SirT1 ASO–treated rats (n = 4/group). *P < 0.05, **P < 0.01 (left). Cytokine array performed on plasma from chow-fed wild-type and fat-specific SirT1 knockout mice (n = 4/group). *P < 0.08, **P < 0.03 (right). B: Plasma IL-10 levels collected at the onset of hypophagia (1 week) (n = 10/group) and plasma IL-4 after 1 month of ASO treatment. C: Representative hematoxylin and eosin staining from epididymal WAT of individual rats fed normal chow and treated with control ASO or SirT1 ASO (representative of 5 rats/group). GM-CSF, granulocyte-macrophage colony-stimulating factor; KO, knockout; N.S., nonsignificant. (A high-quality digital representation of this figure is available in the online issue.)

17. FIG. 2. SirT1 knockdown/deletion causes adipose tissue inflammation and macrophage infiltration. D: Reduced adipocyte diameter in SirT1 ASO–treated rats. E: Adipose tissue macrophage content, as assessed by FACS, and adipose tissue CD68 mRNA expression in mice (n = 5/group). F: Macrophage/monocyte (CD68, CD115) and macrophage/dendritic cell (CD11c) marker mRNA expression in rat adipose tissue (n = 4–7). G: Macrophage-related mRNA abundance in adipose tissue from SirT1 fat-specific knockout mice fed a chow diet (8–12 weeks of age, n = 4–6/group). H: Macrophage marker/cytokine mRNA abundance in adipose tissue from SirT1 fat-specific knockout mice fed a high-fat diet for 1 month (n = 3–7/group). GM-CSF, granulocyte-macrophage colony-stimulating factor; KO, knockout; N.S., nonsignificant. (A high-quality digital representation of this figure is available in the online issue.)

18. FIG. 3. SirT1 knockdown in WAT results in NF-kB nuclear localization and gene expression through reduction of H3K9 deacetylation. A and B: Representative (n = 5/group) images showing that SirT1 knockdown stimulates nuclear translocation of NF-kB in rat WAT. Adipocytes are stained for caveolin (green) and NF-kB p65 nuclear localization sequence (red). Nuclei are stained with DAPI (blue). Inset box is expanded to individual channels in B.1-B.3 to demonstrate NF-kB and DAPI colocalization. Some NF-kB–positive nuclei (arrows) are in adipocytes. Because the nuclear localization sequence is masked in the cytosol, nonnuclear staining is nonspecific (i.e., erythrocyte or other autofluorescence). C: SirT1 knockdown increases abundance of phosphorylated (Ser536) and total p65 in rat WAT nuclear lysates. Line separates noncontiguous lanes from the same blot. (A high-quality digital representation of this figure is available in the online issue.)

19. FIG. 3. SirT1 knockdown in WAT results in NF-kB nuclear localization and gene expression through reduction of H3K9 deacetylation. D: Chow-fed fat-specific SirT1 knockout mice have increased phospho-p65 in WAT ;12 h after stimulation with LPS (50 mg i.p.). E: SirT1 knockdown increases adipose tissue cytokine, complement, and TLR mRNA expression (n = 5–7/group). *P < 0.05, **P < 0.01. F: SirT1 knockdown increases H3K9 acetylation. (A high-quality digital representation of this figure is available in the online issue.)

20. FIG. 4. Overexpression of SirT1 reduces high-fat diet–induced increases in WAT macrophage content. A and B: Reduced F4/80 (A) and CD68 (B) expression in WAT of high-fat diet–fed SirT1-overexpressing mice by qPCR. C: Verification of SirT1 overexpression in WAT (n = 5/group). D: NEMO and CASP8 expression, also in WAT (n = 3/group).

21. Characteristics of Human Subjects (n=45). Gender, race, age, BMI, BMIz and percent body fat of human subjects stratified into non-obese, moderately obese, and severely obese pools.

22. FIG. 5. SirT1 transcript level is inversely correlated with both BMI and adipose tissue macrophage content in humans. A: Control, obese, and severely obese subjects were stratified by BMI. Obese and severely obese subjects have decreased SirT1 expression (B) and increased macrophage content (C) in subcutaneous adipose tissue. Macrophages (CD68+ cells) within an entire section were counted by two independent observers using a light microscope. % macrophages = number of macrophages / number of adipocytes 3 100. Obese and severely obese subjects have elevated HOMA-IR (D). **P < 0.01, *P < 0.05, #P < 0.10. Note: 18 of the 45 subjects examined are also being studied for the contributions of a gene polymorphism to SirT1 gene expression.

23. FIG. 6. SirT1 transcript level is inversely correlated with macrophage markers in human adults. A: Correlation between SirT1 mRNA expression and CD14 (a macrophage/monocyte marker) in adipose tissue of a mixed-weight population of humans. B: Correlation between SirT1 and CD86 mRNA (another monocyte/macrophage marker) in WAT from the same group of subjects. C: Correlation between SirT1 and CX3CL1 (a monocyte chemoattractant) mRNA in WAT from the same group of subjects. FFA, free fatty acid; NLR, NOD-like receptor.

24. FIG. 6. SirT1 transcript level is inversely correlated with macrophage markers in human adults. D–F: SirT1 knockdown increases expression of CX3CL1, CX3CR1, and CD14 in rodent WAT. FFA, free fatty acid; NLR, NOD-like receptor.

25. FIG. 6. SirT1 transcript level is inversely correlated with macrophage markers in human adults. G: Schematic model of SirT1 regulation of inflammation. SirT1 deacetylation of inflammatory gene promoters causes decreased cytokine production in response to stimulation of inflammatory sensors by fatty acids, hypoxia, and ER stress. In turn, decreased SirT1 expression in obesity sensitizes these networks to activation by stressors. FFA, free fatty acid; NLR, NOD-like receptor.

26. RESULTS—We found that inducible or genetic reduction of SirT1 in vivo causes macrophage recruitment to adipose tissue, whereas overexpression of SirT1 prevents adipose tissue macrophage accumulation caused by chronic high-fat feeding. We also found that SirT1 expression in human subcutaneous fat is inversely related to adipose tissue macrophage infiltration.

27. CONCLUSIONS—Reduction of adipose tissue SirT1 expression, which leads to histone hyperacetylation and ectopic inflammatory gene expression, is identified as a key regulatory component of macrophage influx into adipose tissue during overnutrition in rodents and humans. Our results suggest that SirT1 regulates adipose tissue inflammation by controlling the gain of proinflammatory transcription in response to inducers such as fatty acids, hypoxia, and endoplasmic reticulum stress.

28. the 1Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan; and the 2Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan. These findings further support the therapeutic potential of SirT1 activators for the obesity-induced metabolic diseases. In several rodent models of type 2 diabetes, SirT1 activators (e.g., resveratrol) and moderate SirT1 overexpression can ameliorate insulin resistance . The observation that SirT1 is decreased concomitantly with obesity raises questions regarding the approach to take with pharmacologic activation of SirT1. Overall, these findings shed light on another regulator of the “tone” of adipose tissue inflammation that opens the door to new treatment possibilities. DIABETES, VOL. 60, DECEMBER 20113100

29. The vicious cycle of adipose tissue inflammation. During obesity, nutrient excess and microenvironmental alterations lead to free fatty acids and cytokine release that recruits and activates inflammatory macrophages. ATM activation leads to local proinflammatory cytokine production triggered by innate immune sensors (e.g., JNK and the inflammasome). Cytokines talk back to adipocytes and contribute to further adipocyte dysfunction that amplifies inflammatory signals through a feed-forward loop. SirT1 suppresses the proinflammatory gene expression by deacetylating histones in adipocytes and macrophages and disrupting adipocyte-macrophage communication. T cells in fat can influence both ATMs and adipocytes and may be another target for SirT1 action. C3, complement factor 3; Tregs, regulatory T cells. DIABETES, VOL. 60, DECEMBER 20113100

30. Message/Comments カロリー摂取の制限により活性化される長寿遺伝子「SIRT1」 で、 脂肪組織ではSirt1が抑制されるとマクロファージが集まったり炎症促進となる。 ヒトでも肥満でSirt1が減少している。 （Sirt1は良い因子であり活性化させたりするとよいだろう。）