Journal Club
Gillum MP, 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.
2011年12月15日 8:30-8:55
８階 医局
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
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
Resveratrol has been claimed to be an activator of Sirtuin 1,
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.
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 NAD1dependent manner. SIRT1 induces gluconeogenic genes and hepatic glucose output through PGC1a, 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)
1 Sirtris 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.
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 glucosestimulated release of insulin) has been
shown, its effect on metabolism in brain and
skeletal muscle is still elusive
Nature Medicine 12, 34 - 36 (2006)
Figure 1 | Identification of potent SIRT1 activators unrelated to resveratrol.
a, Chemical structures of SIRT1 activators, resveratrol, SRT1460, SRT2183,
and SRT1720.
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 。
http://www.daiwa-grp.jp/dsh/results/33/pdf/08.pdf
FOXO1を刺激し
インスリン抵抗性を
起す？！
PPARγを抑制しイ
ンスリン抵抗性を起
す
血糖上昇
血糖上昇
他に
アポトーシスを抑制
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。
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.
Diabetes 60:3235–3245, 2011
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.
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.
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 Ruslan Medzhitov (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.
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 Qiagen RNeasy kit.
Transcript abundance was assessed by real-time PCR on a 7500 Fast RealTime 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).
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.
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.
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.
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.)
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: Macrophagerelated 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.)
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.)
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.)
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 SirT1overexpressing mice by qPCR. C: Verification of SirT1 overexpression in WAT (n = 5/group). D:
NEMO and CASP8 expression, also in WAT (n = 3/group).
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.
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.
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.
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.
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.
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.
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.
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 2011 3100
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 2011 3100
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。）