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. 2010 Dec 16;468(7326):933-9.
doi: 10.1038/nature09564.

CRTC3 links catecholamine signalling to energy balance

Youngsup Song  1 , Judith Altarejos, Mark O Goodarzi, Hiroshi Inoue, Xiuqing Guo, Rebecca Berdeaux, Jeong-Ho Kim, Jason Goode, Motoyuki Igata, Jose C Paz, Meghan F Hogan, Pankaj K Singh, Naomi Goebel, Lili Vera, Nina Miller, Jinrui Cui, Michelle R Jones; CHARGE Consortium; GIANT Consortium; Yii-Der I Chen, Kent D Taylor, Willa A Hsueh, Jerome I Rotter, Marc Montminy
Affiliations

CRTC3 links catecholamine signalling to energy balance

Youngsup Song et al. Nature. .

Abstract

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 β-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 β-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.

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Figures

Figure 1
Figure 1
CRTC3−/− mice are resistant to diet induced obesity. A. Top, schematic of CRTC3 protein showing CREB Binding (CBD: aa. 1–50), regulatory (RD; aa. 51–549), and transactivation (TAD: aa. 550–619) domains as well as regulatory Ser162 site, which corresponds to a consensus phosphorylation site for AMPK family members. Middle, immunoblot showing effect of FSK on de-phosphorylation of CRTC3 in wild-type and CRTC3−/− mouse embryo fibroblasts (MEFs) exposed to FSK for 1 or 4 hours. Bottom, immunoblot showing nuclear and cytoplasmic amounts of CRTC3 in NIH-3T3L1 pre-adipocytes under basal conditions and following exposure to FSK. B. Effect of FSK on CRE-luciferase reporter activity in HEK293T cells following over-expression of wild-type or phosphorylation-defective S162A CRTC3. Co-transfection of dominant negative A-CREB expression vector indicated. C. Q-PCR (top) and immunoblot (bottom) analysis of CRTC3 mRNA and protein amounts in different tissues in wild-type and CRTC3 −/− (KO) mice. CRTC1 protein amounts shown for comparison. (CTX; cortex. CBM; cerebellum. LIV; liver. WAT, BAT; white and brown adipose. MUS; muscle. PAN; pancreas.) D. Top, schematic showing structure of the CRTC3 targeting vector containing Neo selection marker in place of Exon 1, which encodes the CREB binding domain (CBD). Structure of the mutant CRTC3 allele after homologous recombination indicated. Bottom, PCR analysis showing wild-type and mutant CRTC3 alleles in genomic DNA from wild-type, heterozygous, and homozygous CRTC3 knockout mice. E. Relative weight gain in wild-type and CRTC3 mutant (Het, KO) littermates under normal chow (12% of calories from fat; top) or high fat diet feeding (HFD; 60% of calories from fat; bottom) conditions. Age (in weeks) indicated. (*; P<0.05. **; P<0.01. ***; P <0.001.) F. Left, relative fat and lean mass in HFD-fed wild-type and CRTC3−/− mice by MRI analysis. Right, representative photo of HFD-fed wild-type and CRTC3−/− mice. (*; P<0.05)
Figure 2
Figure 2
Increased energy expenditure and insulin sensitivity in CRTC3−/− mice. A. Relative energy expenditure and oxygen consumption in wild-type and CRTC3−/− mice under high fat diet feeding conditions. B. Metabolic cage analysis of food intake and physical activity in wild-type and CRTC3−/− mice maintained on a HFD for 12 weeks. C. Top, circulating concentrations of free fatty acids (FFAs) in wild-type and CRTC3−/− mice under ad libitum feeding conditions. Bottom, hematoxylin-eosin staining of hepatic sections showing relative amounts of lipid in HFD fed wild-type and CRTC3−/− mice. (**; P<0.01) D. Top, Circulating leptin concentrations in NC and HFD-fed wild-type and CRTC3−/− mice. Bottom, effect of leptin (3mg/kg) or vehicle injection (IP) on energy expenditure in wild-type and CRTC3−/− mice. (*; P<0.05. ***; P<0.001.) E. Top, immunohistochemical analysis of WAT sections from wild-type and CRTC3−/− mice using F4/80 antiserum to visualize resident adipose tissue macrophages. Scale bar, 50μm. Bottom, Q-PCR analysis of mRNA amounts for macrophage-specific genes in WAT from HFD-fed wild-type and CRTC3−/− mice. F. Circulating concentrations of insulin (top), insulin tolerance testing (middle), and glucose tolerance testing (bottom) of wild-type and CRTC3−/− mice maintained a high fat diet (HFD) for 10 weeks. Insulin levels on NC diet (top) shown for comparison. (*; P<0.05. **; P<0.01. ***; P<0.001.)
Figure 3
Figure 3
Increased catecholamine signaling in CRTC3−/− adipose. A. Top, In vivo imaging analysis showing CRE-luc reporter activity in tissues from transgenic reporter mice following IP administration of isoproterenol (10mg/kg). Bar graph shows reporter activity in extracts from BAT, WAT, and liver. Bottom left, immunoblot of CRTC3 protein amounts in WAT from wild-type mice under fasting or fed conditions. Bottom right, immunoblot showing effect of leptin injection IP (3mg/kg; s.i.d. for 3d) on CRTC3 de-phosphorylation in BAT. B. Top, H&E sections of WAT from wild-type and CRTC3−/− mice. Scale bar, 50μm. Bottom, relative size distribution of adipocytes from wild-type and CRTC3 mutant mice. C. Left, in vitro lipolysis rates in cultured wild-type or CRTC3−/− adipocytes exposed to isoproterenol (ISO; 2μM). Right, relative lipolysis rates in wild-type and CRTC3−/− adipocytes exposed to FSK for 1 or 2 hours. Lipolysis rates determined by measuring glycerol release into the medium. (*; P<0.05. **; P<0.01). D. Top, immunoblot analysis of phospho (Ser660) HSL amounts in WAT from NC or HFD-fed mice following IP ISO administration as indicated. Bottom left, immunoblot of P-HSL amounts in WAT from WT or CRTC3−/− mice under HFD conditions. Bottom right, PKA activity, as measured with phospho- PKA substrate antiserum in epididymal WAT from HFD-fed wild-type and CRTC3−/− mice. E. Top, H&E sections of BAT from wild-type and CRTC3−/− mice. Scale bar, 50μm. Bottom, relative numbers of brown adipocytes recovered from fat pads of wild-type and CRTC3 mutant mice. (***; P<0.001). F. Top, in vitro fatty acid oxidation rates in brown adipocytes, normalized to total cell number. Exposure to FSK (10μM) plus isoproterenol (10μM; F/I) indicated. Bottom left, UCP1 mRNA amounts, determined by Q-PCR analysis of BAT mRNA from wild-type and CRTC3−/− mice (**; P<0.01). Bottom right, core body temperatures in wild-type and CRTC3−/− mice..
Figure 4
Figure 4
CRTC3 attenuates cAMP signaling in adipose. A. cAMP content in WAT (top) and cultured MEFs (bottom) from wild-type and CRTC3−/− mice. Exposure to FSK indicated. (**; P<0.01) B. Effect of CRTC3 over-expression (top) or RNAi mediated knockdown (bottom) on cAMP accumulation in wild-type MEFs exposed to FSK as indicated. (***; P<0.001) C. Top, Q-PCR analysis showing relative mRNA amounts for RGS2 in primary cultured adipocytes (WT, CRTC3−/−) exposed to FSK as indicated. Bottom, Q-PCR analysis of RGS2 mRNA amounts in WAT from NC or HFD-fed mice (WT, CRTC3−/−). (*; P<0.05. **; P<0.01) D. Effect of RGS2 over-expression (top) or RNAi mediated knockdown (bottom) on cAMP accumulation in wild-type MEFs exposed to FSK as shown. (*; P<0.05. ***; P<0.001) E. Top, transient assay of RGS2-luc reporter activity in HEK293T cells exposed to FSK. Effect of CRTC3 over-expression or RNAi-mediated depletion indicated. Bottom, chromatin immunoprecipitation (ChIP) assay showing occupancy of CRTC3 over the RGS2 promoter in MEFs exposed to FSK as indicated. F. Top, immunoblot showing relative amounts of wild-type and S72N variant epitope-tagged CRTC3 polypeptides in cytoplasmic and nuclear fractions from transfected HEK293T cells. Fractionation of control cytoplasmic (ACC) and nuclear (CREB) proteins shown. Bottom, transient transfection assay of HEK293T cells showing relative activities of wild-type and S72N CRTC3 expression vectors co-transfected with RGS2-luc reporter plasmid. Exposure to FSK indicated.
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