肥胖、消瘦？-Nature 发现调控能量平衡的基因-----2010-12月

ARTICLE doi:10.1038/nature09564 CRTC3 links catecholamine signalling to energy balance Youngsup Song1, Judith Altarejos1, Mark O. Goodarzi2, Hiroshi Inoue1, Xiuqing Guo3, Rebecca Berdeaux1, Jeong-Ho Kim1, Jason Goode1, Motoyuki Igata1, Jose C. Paz1, Meghan F. Hogan1, Pankaj K. Singh1, Naomi Goebel1, Lili Vera1, Nina Miller1, Jinrui Cui3, Michelle R. Jones2, CHARGE Consortium, GIANT Consortium, Yii-Der I. Chen3, Kent D. Taylor3, Willa A. Hsueh4, Jerome I. Rotter3 & Marc Montminy1* The adipose-derived hormone leptin maintains energy balance in part through central nervous system-mediated increases in sympathetic outflow that enhance fat burning. Triggering of b-adrenergic receptors in adipocytes stimulates energy expenditure by cyclic AMP (cAMP)-dependent increases in lipolysis and fatty-acid oxidation. Although the mechanism is unclear, catecholamine signalling is thought to be disrupted in obesity, leading to the development of insulin resistance. Here we show that the cAMP response element binding (CREB) coactivator Crtc3 promotes obesity by attenuating b-adrenergic receptor signalling in adipose tissue. Crtc3 was activated in response to catecholamine signals, when it reduced adenyl cyclase activity by upregulating the expression of Rgs2, a GTPase-activating protein that also inhibits adenyl cyclase activity. As a common human CRTC3 variant with increased transcriptional activity is associated with adiposity in two distinct Mexican-American cohorts, these results suggest that adipocyte CRTC3 may play a role in the development of obesity in humans. Obesity is a major risk factor in the development of insulin resistance, which is characterized by decreased glucose uptake into muscle and increased glucose production by the liver. Obesity affects one-third of adults in the USA1; the prevalence of obesity appears even higher in certain ethnic groups, although relevant predisposing factors have not been fully identified. Obesity is a particular problem amongMexican- Americans, with an overall prevalence of 40% (36% in men, 45% in women)1, contributing to elevated rates of diabetes2. Environment, lifestyle and genetic susceptibility probably contribute to the increased risk of obesity and diabetes in this population. Under lean conditions, the adipose-derived hormone leptin is thought to promote energy expenditure through increases in sym- pathetic nerve activity that enhance catecholamine signalling in white adipose tissue (WAT) and brown adipose tissue (BAT)3,4. Triggering of b-adrenergic receptors appears important for subsequent increases in lipolysis and fatty-acid oxidation5; mice with knockouts of all three b receptors have reduced energy expenditure, and they are more susceptible to effects of high-fat diet (HFD) feeding on weight gain. Conversely, transgenic over-expression of b-adrenergic receptor 1 in adipose tissue appears sufficient to confer resistance to obesity6. Triggering of b-adrenergic receptors stimulates cAMP-mediated increases in cellular gene expression with burst-attenuation kinetics7,8; rates of transcription peak within 1 h of stimulation and decrease thereafter even under continuous stimulation. Although the under- lying mechanisms remain unclear, the attenuation of cellular genes is thought to be coordinated by negative feedback effectors, which are themselves targets for upregulation by cAMP9. cAMPstimulates the expression of cellular genes through the protein kinase A (PKA)-mediated phosphorylation of CREB family members (CREB1, ATF1, CREM), a modification that promotes recruitment of the histone acetyl transferase paralogues P300 and CBP10–12. In parallel, cAMP also increases gene expression by stimulating the CREB regulated transcriptional coactivators (CRTCs)13,14. Under basal conditions, CRTCs are sequestered in the cytoplasm through phos- phorylation-dependent interactions with 14-3-3 proteins. CRTCs are phosphorylated by salt-inducible kinases and other members of the stress- and energy-sensing AMPK family of Ser/Thr kinases. Increases in intracellular cAMP signalling promote the PKA-mediated phos- phorylation and inhibition of salt-inducible kinase activity, leading to the subsequent dephosphorylation and nuclear entry of CRTCs, which bind to CREB over relevant promoters. After prolonged stimulation with cAMP agonist, CRTC activity is terminated through ubiquitin-mediated degradation15. TheCRTC family consists of threemembers (Crtc1,Crtc2 andCrtc3), which are distinguished in part by their expression profiles. Crtc1 is produced primarily in brain, where itmediates leptin effects on satiety16; mice with a knockout of the Crtc1 gene develop obesity due in part to reductions in energy expenditure. By contrast, Crtc2 is expressed at high levels in liver where it promotes fasting gluconeogenesis17,18; mice with a knockout ofCrtc2 appearmore insulin sensitive underHFD conditions, owing to reductions in hepatic glucose output19. Role of CRTC3 as a CREB coactivator Similar to other CRTC family members, Crtc3 contains CREB binding (CBD; amino acids 1–50), regulatory (RD; amino acids 51–549) and trans-activation domains (TAD; amino acids 550–619), which are also present in Crtc1 and Crtc2 (Fig. 1a). In the basal state, Crtc3 is phos- phorylated at Ser 162 by salt-inducible kinases and other members of the stress- and energy-sensing AMPK family of Ser/Thr kinases13,20,21. Short-term (0.5–1 h) exposure to cAMP agonist promotes the depho- sphorylation and nuclear entry of Crtc3 (Fig. 1a); similar to Crtc215, prolonged cAMP stimulation triggers Crtc3 degradation. Crtc3 over-expression augments the activity of a cAMP responsive (CRE-luc) reporter in cells exposed to forskolin (FSK; Fig. 1b); and mutation of the regulatory Ser162 phosphorylation site to alanine fur- ther enhancesCrtc3 activity under basal conditions. In keepingwith the 1The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA. 2Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA. 3Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA. 4Diabetes Research Center, Division of Diabetes, Obesity and Lipids, Methodist Hospital Research Institute, Houston, Texas 77030, USA. 1 6 D E C E M B E R 2 0 1 0 | V O L 4 6 8 | N A T U R E | 9 3 3 Macmillan Publishers Limited. All rights reserved©2010 proposed role of CREB in recruiting CRTC3 to relevant promoters, expression of a dominant negative CREB inhibitor, called ACREB22, blocks Crtc3 effects on reporter activity in cells exposed to FSK. By contrastwithCrtc1,which is expressed primarily inbrain,Crtc3 protein and messenger RNA (mRNA) amounts are particularly abundant in WAT and to a lesser extent in BAT (Supplementary Fig. 1 and Fig. 1c). 0 100 200 300 DMSO FSK Crtc3 Crtc3-S162A ACREB − − − − − − −− − − − - + + + + + + + C R E -l uc a ct iv ity a b c d e f Neo PgkDTA Exon1 1.5 kb0.8 kb 6.89 kb3.33 kb Exon2 Exon2Neo WT allele Targeting vector Crtc3−/− allele CBD RD TAD 1 50 550 619 CRTC3 : 162 φxBTSxxxϕ LNRTSDSAL W T K O W T K O W T K O W T K O W T K O W T K O W T K O 0 5,000 10,000 15,000 20,000 25,000 WT C R TC 3/ G A P D H C TX C B M LI V W A T B A T M U S P A N CRTC3 +/+ +/− −/− Crtc3−/− allele WT allele 19 Normal chow 3 5 7 9 11 13 15 17 10 15 20 25 30 35 WT Crtc3+/− Crtc3−/− Crtc3+/− Crtc3−/− * * Age (weeks) W ei gh t (g ) 60% HFD 7 9 11 13 15 17 19 15 20 25 30 35 40 45 50 WT ** *** *** *** *** Age (weeks) W ei gh t (g ) WT Crtc3−/− CYT NUC FSK (Hr) − 0.5 1 − Crtc3 Tub 0.5 1 0 10 20 WT Crtc3−/− Crtc3−/− Fa t m as s (g ) * 0 10 20 30 Le an m as s (g ) Hsp90 WT KO WT KO WT KO WT KO FSK 1 h FSK 4 h pCrtc3 Crtc3 CBD Crtc3 Crtc1 Hsp90 Figure 1 | Crtc32/2 mice are resistant to obesity. a, Top, CREB-binding (CBD), regulatory (RD) and transactivation (TAD) domains and conserved AMPK/salt-inducible kinase phosphorylation site (Ser 162). Consensus phosphorylation site for AMPK familymembers (QxBTSxxxQ) shown; relative position of hydrophobic (Q), basic (B), Thr (T), and phosphorylated Ser (S) residues indicated. x represents any amino acid. Middle, immunoblot of Crtc3 in wild-type (WT) and Crtc32/2 (knockout, KO) MEFs exposed to FSK. Bottom, effect of FSK on nuclear and cytoplasmic Crtc3 levels. b,Effect of wild- type or S162A Crtc3 on CRE-luciferase activity. c,Quantitative PCR (top) and immunoblot (bottom) analysis of Crtc3 tissue expression. BAT, brown adipose; CBM, cerebellum; CTX, cortex; LIV, liver; MUS, skeletal muscle; PAN, pancreas; WAT, white adipose. d, Top, Crtc3 targeting vector with Neo selection marker replacing Exon 1, which encodes the CBD. Bottom, PCR analysis of wild-type and mutant Crtc3 alleles in mice. e, Weight gain in wild- type and Crtc3mutant mice maintained on normal chow (n58 per group) or HFD (n55) (*P,0.05; **P,0.01; ***P,0.001.). f, Fat mass (left) and photograph (right) of HFD-fed wild-type and Crtc32/2 mice (n54 per group) (*P,0.05). Error bars, s.e.m. RESEARCH ARTICLE 9 3 4 | N A T U R E | V O L 4 6 8 | 1 6 D E C E M B E R 2 0 1 0 Macmillan Publishers Limited. All rights reserved©2010 Based on the importance of the CBD for Crtc-mediated induction of cAMP-responsive genes23,24, we generated Crtc32/2 mice with a deletion of exon 1, which encodes the CBD (Fig. 1d). Crtc32/2 mice are born at the expected Mendelian frequency; they appear com- parable to wild-type littermates at birth, despite the absence of detect- able Crtc3 mRNA and protein amounts in all tissues (Fig. 1c). Role of CRTC3 in energy balance Whenmaintained on a normal chowdiet,Crtc32/2mice appearmore insulin sensitive than controls by insulin tolerance testing (Sup- plementary Fig. 1, right).Crtc32/2 animals also have 50% lower adipose tissue mass, despite comparable food intake and physical activity to control mice (Supplementary Fig. 2). When transferred to anHFD(60%of calories fromfat),Crtc32/2mice gained35% lessweight relative to controls reflectingprimarily differences in fat accumulation (Fig. 1e, f). The effect of Crtc3 on adiposity appeared to be dependent on gene dosage as Crtc31/2 mice show intermediate weight gains relative to wild-type and Crtc32/2 mice. Although physical activity and food intake were nearly identical, energy expenditure and oxygen consumption were substantially elevated in HFD-fed Crtc32/2 mice relative to wild-type littermates (Fig. 2a, b). Pointing to parallel increases in glucose and lipid oxidation, respiratory quotients were com- parable in wild-type and Crtc32/2 mice (Supplementary Fig.3). Circulating concentrations of free fatty acids were decreased in Crtc32/2 mice, and they were protected from the effects of HFD feeding on hepatic steatosis (Fig. 2c). Consistent with their reduced fat mass, Crtc32/2 mice had decreased circulating leptin concentra- tions compared with wild-type littermates, although the reduction in leptin levels (tenfold) appeared disproportionately low relative to the difference in fat mass (threefold) (Fig. 2d and Supplementary Fig. 4). Indeed, intraperitoneal administration of leptin stimulated energy expenditure to a greater extent in Crtc3 mutant than wild-type mice. Taken together, these results indicate that disruption of Crtc3 activity leads to increases in energy expenditure, which maintain leptin sensi- tivity and protect against ectopic lipid accumulation. Under obese conditions, increases in inflammatory infiltrates in adipose tissue contribute to the development of systemic insulin res- istance25. Although they were readily observed in wild-type mice, adipose-tissue macrophages were less abundant in Crtc32/2 tissue (Fig. 2e and Supplementary Fig. 5). Arguing against an effect of the Crtc3 knockout on macrophage function per se, tumour necrosis factor-a release from peritoneal macrophages in response to lipopo- lysaccharide appeared comparable between Crtc3mutant and control cells (Supplementary Fig. 5). In line with these differences, circulating insulin concentrations were lower in HFD-fed Crtc32/2 than wild- type mice, and whole-body insulin sensitivity was correspondingly improved by insulin and glucose tolerance testing (Fig. 2f). As a result, glucose uptake intomuscle was increased inCrtc32/2 mice compared with control littermates (Supplementary Fig. 6). We considered that Crtc3 activity in adipose tissue may be modu- lated by hormonal signals. In line with its effects on Crtc3 depho- sphorylation in cell cultures (Supplementary Fig. 7), intraperitoneal administration of b-adrenergic agonist isoproterenol (ISO) increased the activity of a CRE-luc reporter transgene in WAT and BAT by live imaging analysis (Fig. 3a). Leptin administration (intraperitoneal) also promoted Crtc3 dephosphorylation. Crtc3 protein amounts in WAT are elevated under ad libitum conditions; they decreased after fasting for 6 , when Crtc3 appeared to undergo degradation (Fig. 3a). Consistent with an increase in protein stability under obese condi- tions, Creb and Crtc3 protein amounts were upregulated in WAT from HFD-fed mice compared with those fed on normal chow (Supplementary Fig. 8). a c e d b 0 20 40 60 WT Crtc3−/− * ***L ep tin (n g m l− 1 ) Normal chow HFD 10 15 20 WT Crtc3−/− H (k ca l h − 1 kg − 1 ) H (k ca l h − 1 kg − 1 ) 2,000 2,500 3,000 3,500 4,000 WT Crtc3−/− VO 2 (m l h − 1 kg − 1 ) WT Crtc3−/− 10 12 14 16 *** *** * Control Leptin 0.0 2.5 5.0 7.5 10.0 *In su lin (μ g l− 1 ) Normal chow HFD 2.0 * 0.0 0.5 1.0 1.5 0 50 100 150 200 WT Crtc3−/− * ** Time (h) B lo od g lu co se (m g dl − 1 ) B lo od g lu co se (m g dl − 1 ) Insulin tolerance testing 0.0 0.1 0.2 0.3 0.4 0.5 WT Crtc3−/− ** FF A (m m ol l− 1 ) 0.0 0.2 0.4 0.6 0.8 1.0 WT Crtc3−/− Ccl3 F4/80 Tlr7 Cd11b m R N A (n or m al iz ed ) WT Crtc3−/− f 2.5 ** 0.0 0.5 1.0 1.5 2.0 0 100 200 300 400 500 WT Crtc3−/− * * *** *** ** Time (h) Glucose tolerance testing WT Crtc3−/− Crtc3−/− WT 0 1 2 3 4 Fo od in ta ke (g d ay − 1 ) WT Crtc3−/− 0 1,000 2,000 3,000 4,000 To ta l a ct iv ity WT Crtc3−/− Figure 2 | Increased energy expenditure in Crtc32/2 mice. a, b, Energy expenditure and oxygen consumption (a) as well as food intake and physical activity (b) inHFD-fedmice (n54 per group). c, Free-fatty-acid levels (top) and haematoxylin and eosin sections of livers (bottom) in HFD-fed mice (n53 per group) (**P,0.01). d, Leptin levels (top) (n55 per group) and effect of intraperitoneal leptin administration on energy expenditure (bottom) (n54 per group) (*P,0.05; ***P,0.001). e, Macrophage infiltration (top) and gene expression (bottom) in WAT from HFD-fed mice. Scale bar, 50mm. f, Insulin levels (top), insulin tolerance testing (middle) and glucose tolerance testing (bottom) of HFD-fed mice (n55 per group) (*P,0.05; **P,0.01; ***P,0.001). Error bars, s.e.m. ARTICLE RESEARCH 1 6 D E C E M B E R 2 0 1 0 | V O L 4 6 8 | N A T U R E | 9 3 5 Macmillan Publishers Limited. All rights reserved©2010 Catecholamine signalling in adipose tissue Under HFD feeding conditions, increases in catecholamine signalling maintain energy balance by mobilizing triglyceride stores in WAT26. Although the total number of adipocytes in WAT fat pads was nearly identical in both groups, adipocytes from Crtc32/2 mice were sub- stantially smaller than from wild-type mice (Fig. 3b). Arguing against a disruption in triglyceride synthesis, mRNA amounts for lipogenic genes (Acc, Lpl, Scd) appeared comparable between Crtc3mutant and wild-type adipocytes (Supplementary Fig. 9). Rather, basal and ISO- induced lipolysis rates were increased in Crtc32/2 compared with control adipocytes (Fig. 3c). Exposure to FSK also increased lipolysis to a greater extent in Crtc32/2 adipocytes (Fig. 3c), pointing to the potential upregulation of the cAMP signalling pathway in these cells. Triggering of b-adrenergic receptors has been found to promote lipolysis through the cAMP-dependent PKA-mediated phosphoryla- tion of hormone sensitive lipase (HSL)27. In keeping with the pro- posed downregulation of b-adrenergic receptor signalling in obesity, administration of ISO had only modest effects on HSL phosphoryla- tion in HFD-fed relative to animals fed on normal chow (Fig. 3d). Indeed, amounts of phospho- (Ser 660)HSLwere substantially elevated ba c d e 0 100 200 300 400 500 B A T ce ll nu m b er s ***WT Fast Ad lib. Hsp90 Creb Crtc3 WT WT Hsp90 pHsl WT pPKA SUB Hsp90 Diameter (μM) 20 40 60 80 100 120 140 160 0 10 20 30 WT A d ip oc yt es (% ) 0 250 500 750 1,000 Control ISO LIV WATBAT R LU 2,650 0 1 2 0 100 200 WT * FSK (h) G ly ce ro l r el ea se (μ M ) WT LIV WAT Control ISO Veh ISO WT P = 0.054 ** G ly ce ro l r el ea se (μ M ) pCrtc3 Crtc3 Hsp90 Control Leptin BAT HSL pHSL Veh ISO Veh ISO NC HFD 300 200 100 0 Crtc3−/− Crtc3−/− Crtc3−/− Crtc3−/− Crtc3−/− Crtc3−/− Crtc3−/− Crtc3−/− Crtc3−/− Crtc3−/− Crtc3−/− f 0 1 2 WT U cp 1 /L 32 34 35 36 WT ** Te m pe ra tu re (º C ) 0 10 20 30 40 WT F/I FA O (p m ol m in − 1 ) Control ** Figure 3 | Increased catecholamine signalling in Crtc32/2 adipose tissue. a, Top, effect of ISO on CRE-luc reporter activity in different tissues. Bottom, immunoblots of Crtc3 in WAT (left) or BAT (right). Effect of fasting (Fast), feeding ad libitum (Ad lib.) and leptin administration indicated. LIV, liver. b, Haematoxylin and eosin sections (top) and adipocyte size distribution (bottom) in WAT from wild-type and Crtc32/2 mice. Scale bar, 50mm. c, Lipolysis rates in adipocytes exposed to ISO (left) or FSK (right) (n53) (*P,0.05; **P,0.01).Veh, vehicle. d, Phospho- (Ser 660) HSL levels in WAT from mice fed on normal chow or HFD after ISO injection (top) and from HFD-fed wild-type or Crtc32/2 mice (bottom left). Bottom right, immunoblot of PKA activity in WAT from HFD-fed mice. e, Haematoxylin and eosin sections (top) and brown adipocyte numbers (bottom) in wild-type and Crtc32/2 BAT. Scale bar, 50mm (***P,0.001). f, Top, fatty-acid oxidation (FAO) and Ucp1 mRNA levels in brown adipocytes. Core body temperatures indicated (n54 per group) (**P,0.01). Error bars, s.e.m. F/I, exposure to forskolin plus isoproterenol. RESEARCH ARTICLE 9 3 6 | N A T U R E | V O L 4 6 8 | 1 6 D E C E M B E R 2 0 1 0 Macmillan Publishers Limited. All rights reserved©2010 in Crtc32/2 WAT compared with wild type, even though circulating concentrations of noradrenaline and adrenaline were similar between the two groups (Fig 3d and Supplementary Fig. 10). PKA activity in WATwas also increased inCrtc32/2mice by immunoblot assay using a phospho-specific PKA substrate antiserum (Fig. 3d). Consistent with the predominant expression of Crtc3 in adipose tissue, PKA activity in other tissues appeared similar between wild-type and Crtc32/2 mice (Supplementary Fig. 11). Having seen that lipolysis rates are increased inWAT, and realizing that circulating free-fatty-acid concentrations are reduced in Crtc3 mutant mice, we considered that fatty-acid oxidation should also be upregulated in this setting. Under HFD conditions, leptin has been proposed to trigger catecholamine-mediated increases in fat burning in BAT, a process known as diet-induced thermogenesis28,29. In keeping with the ability for catecholamines to stimulate BAT expansion, brown adipocyte numbers were increased twofold in intra-scapular fat pads from Crtc32/2 mice compared with controls (Fig. 3e). Suggesting a parallel increase in fat burning, Crtc32/2 brown adipocytes also had smaller intracellular lipid vacuoles than wild-type cells. Moreove