Abstract
Adipocytes increase energy expenditure in response to prolonged sympathetic activation via persistent expression of uncoupling protein 1 (UCP1)1,2. Here we report that the regulation of glycogen metabolism by catecholamines is critical for UCP1 expression. Chronic β-adrenergic activation leads to increased glycogen accumulation in adipocytes expressing UCP1. Adipocyte-specific deletion of a scaffolding protein, protein targeting to glycogen (PTG), reduces glycogen levels in beige adipocytes, attenuating UCP1 expression and responsiveness to cold or β-adrenergic receptor-stimulated weight loss in obese mice. Unexpectedly, we observed that glycogen synthesis and degradation are increased in response to catecholamines, and that glycogen turnover is required to produce reactive oxygen species leading to the activation of p38 MAPK, which drives UCP1 expression. Thus, glycogen has a key regulatory role in adipocytes, linking glucose metabolism to thermogenesis.
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Data availability
RNA-seq data reported in this paper have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive48 database under BioProject PRJNA752350.
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Acknowledgements
We thank members of Saltiel laboratory at UCSD for helpful discussions, comments and suggestions and the UCSD histology core for tissue sectioning and staining. The authors are supported by 1R01 DK117850 and 1R01 HL147883 (A.J.L.); American Diabetes Association no. 1-19-PDF-177 (M.A.-O.); American Diabetes Association grant no. 1-19-JDF-012 (S.M.R.); F32DK124947 (J.M.V.); DK057978 and DK120480 (R.M.E.); R01DK117551, R01DK125820 and R01DK076906 (A.R.S.); and P30 DK063491 (R.M.E. and A.R.S.). E.T.C. is supported by Claudia Adams Barr Program, the Lavine Family Fund and NIH DK123095. H.X. is supported by a postdoctoral fellowship from the National Cancer Center.
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O.K. performed experimental design, experiments, data acquisition and interpretation, and wrote the manuscript. J.M.V. performed experimental design, analysis of data from human studies and wrote the manuscript. H.X. and E.T.C. performed and analysed the global assessment of cysteine oxidation. S.K.M. performed electron microscopy experiments and image acquisition and provided critical edits to the text. S.M.R. performed experimental design and provided critical edits to the text. M.A.-O. performed experiments and interpretation. J.H.D, B.D., L.C. and A.P. performed experiments. R.T.Y., Y.D., C.L., M.D. and R.M.E. performed and analysed RNA-seq experiments. A.J.L. and M.L. provided human gene expression data from the METSIM study and provided edits to the text. M.R. provided human gene expression data and provided critical edits to the text. A.R.S. conceptualized the study, performed data interpretation and wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Glycogen metabolism is enhanced in beige adipocytes.
a, b, Gene expression in iWAT from mice treated with either vehicle or CL-316,243 for. 7 days. n = 5 mice (vehicle) and 6 mice (CL-316,243). c, Protein expression in mice treated as in a, n = 5 mice (vehicle) and 6 mice (CL-316,243). d, Glycogen levels in iWAT of vehicle or CL-316,243 treated mice, n = 4 mice (vehicle) and 5 mice (CL-316,243). e, Electron micrographs of iWAT from vehicle or CL-316,243 treated mice. Arrows point to glycogen granules. Scale bar, Left two images − 1 µm, right two images – 260 nm. Shown are representative images of tissues from 6 different mice (3 vehicle and 3 Cl-316,243). f, Periodic acid-Schiff (PAS) staining for glycogen and UCP1 immunostaining in iWAT of vehicle or CL-316,243 treated miceUcp1. Right panels show a higher magnification of areas marked by a square. Scale bars, 4 left images – 2 mm, high magnification images – 100µm. Shown are representative images of tissues from 6 different mice (3 vehicle and 3 Cl-316,243). g, Gene expression in iWAT-derived stromal-vascular fraction and primary mature adipocytes from vehicle or CL-316,243 treated mice n = 4 biological replicates per treatment. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. #P < 0.05, ##P < 0.01. *Significance between vehicle and CL-316,243 treatment. #Significance between SVF and vehicle treated mature adipocytes. Statistical significance for a, b and d was determined by two-sided t-test, two-way ANOVA with adjustments for multiple comparisons was used for g.
Extended Data Fig. 2 CL-316,243 treatment does not affect the expression of glycogen metabolizing genes in BAT.
a, gene expression in human preadipocytes, differentiated adipocytes or differentiated adipocytes treated with forskolin for 3 days. n = 52 biological replicates per treatment. Statistical significance was determined using two-way ANOVA with adjustments for multiple comparisons, ***-p < 0.001. b, Mice were treated with either vehicle or CL-316,243 for 7 days. Gene expression in BAT was determined using qPCR, n = 5 mice per treatment. c, Quantification of protein expression data shown in Extended Data Fig. 1C (main text), expression was normalized to HSP90. n = 5 mice (Vehicle), n = 6 mice (CL-316,243). Statistical significance was determined using two-sided t-test. d, Protein expression in BAT from mice treated as in b was determined by SDS-PAGE, n = 5 mice (vehicle), n = 6 mice (CL-316,243). No statistical significance detected. Data in a–c are presented as mean ± s.e.m.
Extended Data Fig. 3 PTG-KO does not affect the response to CL-316,243.
a-e, RNAseq data from iWAT-derived mature adipocytes of WT and PTG-KO mice treated with either vehicle or CL-316,243, n =3. a, Differential gene expression analyses of RNAseq data. b, Log2 of fold change of gene expression of CL-316,243 treated WT mice versus vehicle-treated WT mice. Statistical significance was determined using two-sided t-test. c, Log2 of fold change of gene expression of CL-316,243 treated PTG-KO mice versus vehicle treated PTG-KO mice. Statistical significance was determined using two-sided t-test. d, e, Pathway analyses of RNAseq were conducted using Gene Set Enrichment Analysis. f, Quantification of UCP1 protein expression data shown in Fig. 1f (main text), expression was normalized to RalA. n = 4 mice per treatment. Statistical significance was determined using two-sided t-test. g, Serum FFA levels in WT and PTG-KO mice treated with either vehicle or CL-316,243 for 20 min, n = 5 mice per genotype per treatment. Statistical significance was determined using two-way ANOVA with adjustments for multiple comparisons. Data are presented as mean ± s.e.m. *-p < 0.05. ***-p < 0.001. Accession number to cite these SRA data: PRJNA752350.
Extended Data Fig. 4 Adipose specific PTG-KO reduces energy expenditure.
a, Quantification of protein expression data shown in Fig. 2a (main text), expression was normalized to HSP90. n = 5 mice per treatment per genotype. b, c, Carbon dioxide production (VCO2) in WT and PTG-AKO mice treated with CL-316,243. n = 4 mice per treatment per genotype. b, Average VCO2 over the first three days of CL-316,243 treatment. c, Average VCO2 during days 4-7 of CL-316,243 treatment. d, Quantification of UCP1 protein expression data shown in Fig. 2e (main text), expression was normalized to HSP90. n = 4 mice per treatment per genotype. e, Gene expression in iWAT of WT and PTG-AKO mice fed HFD for three months and then treated with either vehicle or CL-316,243 for 7 days. n = 6 mice per treatment per genotype. f, Body weight of WT and PTG-AKO mice fed HFD for three months before (day 0) and after 7 days (day 6) daily injections of Cl-316,243. n = 7 mice (WT), n = 6 mice (PTG-AKO). g, Weight of the inguinal white adipose tissue of WT and PTG-AKO mice fed HFD for three months and then treated for 7 days with either vehicle or Cl-316,243. n = 7 mice (WT), n = 6 mice (PTG-AKO). h, Linear regression analysis on anthropometric measurements in relation to gene expression from adipose tissue of 770 men. Data are presented as mean ± s.e.m. Statistical significance determined by two-way ANOVA with adjustments for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001. #P < 0.05. * - significance between vehicle and Cl-316-243 treatment within the same genotype. #- significance between WT and PTG-AKO mice treated with CL-316,243.
Extended Data Fig. 5 Overexpression of PTG increases UCP1 expression in vitro.
a, Glycogen levels in mouse primary hepatocytes treated with either vehicle or glucagon (10nM). n = 2 biological replicates per treatment. b, c, HEK-293t cells transfected with a Ucp1-promoter-driven turbo-GFP (Ucp1-GFP) alone or with FLAG tagged PTG (PTG-FLAG). b, Images of transfected cells were acquired using the Nikon eclipse Ts2R microscope. Shown are representative images from 3 independent experiments. Scale bar − 1µm. c, GFP expression levels were determined by western blot. d, Preadipocytes were treated with either Cl-316,243 alone or in combination with Tautomycin. Protein expression was determined by western blot. n = 3 biological replicates per treatment. e, Quantification of protein expression data shown in D. n = 3 biological replicates per treatment. f, Quantification of protein expression data shown in Fig. 3e (main text). n = 2 biological replicates per treatment per genotype. g, Quantification of protein expression data shown in Fig. 3g (main text). n =3 biological replicates per treatment. Data are presented as mean ± s.e.m. Statistical significance was determined using two-way ANOVA with adjustments for multiple comparisons. *P < 0.05, **P < 0.01. #P < 0.05. * - Significance between the zero time point and Glucagon/Cl-316,243 treatment within the same genotype. # - Significance between genotypes within the same time point.
Extended Data Fig. 6 Glycogen metabolism affects ROS production in response to Cl-316,243.
a, Quantification of protein expression data shown in Fig. 4c (main text). n = 2 biological replicates per treatment. Data are presented as mean ± s.e.m. Statistical significance was determined using two-way ANOVA with adjustments for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001. #P < 0.05. * - Significance between the zero time point and the assigned time point within the same treatment. # - Significance between different vehicle and NAC treatments within the same time point. b and c, Global cysteine oxidation in preadipocytes treated with either vehicle or Cl-316,243 (b) or Cl-316,243 alone or in combination with GPI (c), was determined as described in the methods section. b, Blue dots represent proteins with higher cysteine oxidation in vehicle-treated cells, red dots represent proteins with higher cysteine oxidation in Cl-316,243 treated cells. c, Blue dots represent proteins with higher cysteine oxidation in Cl-316,243 treated cells, red dots represent proteins with higher cysteine oxidation in cells treated with Cl-316,243 in combination with GPI. Statistical significance was determined using two-sided t test, cysteine sites shown had at least 5% change between treatments with a p < 0.05.
Extended Data Fig. 7 PTG-KO reduces energy expenditure during long term cold adaptation.
a, Core body temperature of WT and PTG-KO mice measured every 90 min during an acute cold exposure (4 oC), n = 8 mice per genotype. b, Brown adipose tissue glycogen levels in WT and PTG-KO mice at room temperature (RT) or cold exposed for 6 h and allowed to recover for 4 h at RT (4 oC-RT), n = 3 mice per genotype per condition. c, Core body temperature during a second cold exposure in WT and PTG-KO mice that were first cold exposed for 6 h and allowed to recover at room temperature for 4 h, n = 8 mice per genotype. d, e and f, gene expression in iWAT of WT and PTG-BKO mice housed either at room temperature or subjected to prolonged cold adaptation (7 days at 18 oC followed by 14 days at 4 oC), n = 8 mice per genotype. g, Protein expression in iWAT of the mice described in d, n = 5 mice per genotype. h, Oxygen consumption (VO2) in WT and PTG-BKO mice before and during prolonged cold adaptation n = 8 mice per genotype. i, Working model. Acute activation of the β3-adrenergic receptor in white adipocytes results in lipolysis and glycogenolysis through the activation of PKA and subsequent activation of HSL and GP respectively. Prolonged β3-adrenergic receptor activation results in enhanced glycogen accumulation turnover which is required for ROS-dependent p38 activation and the subsequent expression of Ucp1. Data are presented as mean ± s.e.m. Statistical significance was determined two-way ANOVA with adjustments for multiple comparisons. *P < 0.05, **P < 0.01. #P < 0.05. * - significance between room temperature and cold exposure within the same genotype. #- significance between WT and PTG-KO/PTG-BKO with the same treatment.
Extended Data Fig. 8 Increased expression of BAT-Ucp1 following long-term cold exposure does not require PTG.
a, Quantification of protein expression data shown in Extended Data Fig. 7 (main text). n = 5 mice per genotype. b, Protein expression levels in BAT of WT and PTG-BKO mice housed either at room temperature or subjected to prolonged cold adaptation was determined by SDS-PAGE, n (22 oC) = 4, n (4 oC) = 5 . c carbon dioxide production (VCO2) in WT and PTG-BKO mice before and during prolonged cold adaptation. n = 8 mice per genotype. Data are presented as mean ± s.e.m. Statistical significance was determined using two-sided t test (A) or two way ANOVA with adjustments for multiple comparisons (C). *P < 0.05. #P < 0.05. * - Significance between 4 oC and 22 oC within the same genotype. # - Significance between different genotypes within the same time point.
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Supplementary Table 1
This file contains the qPCR primers used in the study.
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Keinan, O., Valentine, J.M., Xiao, H. et al. Glycogen metabolism links glucose homeostasis to thermogenesis in adipocytes. Nature 599, 296–301 (2021). https://doi.org/10.1038/s41586-021-04019-8
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DOI: https://doi.org/10.1038/s41586-021-04019-8
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