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Original article

Adipose tissue glycogen accumulation is
associated with obesity-linked inflammation in
humans

Victòria Ceperuelo-Mallafré 1, 2, 11, Miriam Ejarque 1, 2, 11, Carolina Serena 1, 2, Xavier Duran 2,
Marta Montori-Grau 2, 3, Miguel Angel Rodríguez 2, 4, Oscar Yanes 2, 5, Catalina Núñez-Roa 1, 2, Kelly Roche 1, 2,
Prasanth Puthanveetil 6, Lourdes Garrido-Sánchez 7, 8, Enrique Saez 9, Francisco J. Tinahones 7, 8,
Pablo M. Garcia-Roves 10, Anna Ma Gómez-Foix 2, 3, y, Alan R. Saltiel 6, Joan Vendrell 1, 2, *, 12,
Sonia Fernández-Veledo 1, 2, *, 12

ABSTRACT

Objective: Glycogen metabolism has emerged as a mediator in the control of energy homeostasis and studies in murine models reveal that
adipose tissue might contain glycogen stores. Here we investigated the physio(patho)logical role of glycogen in human adipose tissue in the
context of obesity and insulin resistance.
Methods: We studied glucose metabolic flux of hypoxic human adipoctyes by nuclear magnetic resonance and mass spectrometry-based
metabolic approaches. Glycogen synthesis and glycogen content in response to hypoxia was analyzed in human adipocytes and macro-
phages. To explore the metabolic effects of enforced glycogen deposition in adipocytes and macrophages, we overexpressed PTG, the only
glycogen-associated regulatory subunit (PP1-GTS) reported in murine adipocytes. Adipose tissue gene expression analysis was performed on wild
type and homozygous PTG KO male mice. Finally, glycogen metabolism gene expression and glycogen accumulation was analyzed in adipose
tissue, mature adipocytes and resident macrophages from lean and obese subjects with different degrees of insulin resistance in 2 independent
cohorts.
Results: We show that hypoxia modulates glucose metabolic flux in human adipocytes and macrophages and promotes glycogenesis. Enforced
glycogen deposition by overexpression of PTG re-orients adipocyte secretion to a pro-inflammatory response linked to insulin resistance and
monocyte/lymphocyte migration. Furthermore, glycogen accumulation is associated with inhibition of mTORC1 signaling and increased basal
autophagy flux, correlating with greater leptin release in glycogen-loaded adipocytes. PTG-KO mice have reduced expression of key inflammatory
genes in adipose tissue and PTG overexpression in M0 macrophages induces a pro-inflammatory and glycolytic M1 phenotype. Increased
glycogen synthase expression correlates with glycogen deposition in subcutaneous adipose tissue of obese patients. Glycogen content in
subcutaneous mature adipocytes is associated with BMI and leptin expression.
Conclusion: Our data establish glycogen mishandling in adipose tissue as a potential key feature of inflammatory-related metabolic stress in
human obesity.
Ó 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords Glycogen; Adipocyte; Macrophage; Autophagy; Obesity; Insulin resistance

1
Hospital Universitari de Tarragona Joan XXIII, Institut d’Investigació Sanitària Pere Virgili, Universitat Rovira i Virgili, Tarragona, Spain 2CIBER de Diabetes y Enfermedades
Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain 3Departament de Bioquímica i Biologia Molecular, Institut de Biomedicina de la Universitat de
Barcelona (IBUB), Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain 4Centre for Omic Sciences (COS), Institut d’Investigació Sanitària Pere Virgili, Universitat
Rovira i Virgili, Tarragona, Spain 5Department of Electronic Engineering, Universitat Rovira i Virgili, Tarragona, Spain 6Life Sciences Institute, University of Michigan, Ann
Arbor, MI, USA 7Hospital Universitario Virgen de la Victoria, Instituto de Investigaciones Biomédicas de Málaga (IBIMA), Universidad de Málaga, IBIMA, Spain 8CIBER de
Fisiopatología de Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain 9Department of Chemical Physiology and The Skaggs Institute for Chemical
Biology, The Scripps Research Institute, La Jolla, CA, USA 10Departamento de Ciencias Fisiológicas II, Facultad de Medicina, Universitat de Barcelona, Barcelona, Spain

11
Co-first authors.
12
Co-senior authors.
y
Deceased.

*Corresponding authors. Research Unit, University Hospital of Tarragona Joan XXIII, c/o Dr. Mallafré Guasch, 4, 43007 Tarragona. Spain. Tel.: þ34 977 29 58 00; fax: þ34
977 29 58 23. E-mails: jvortega2002@gmail.com (J. Vendrell), sonia.fernandezveledo@gmail.com (S. Fernández-Veledo).

Received September 17, 2015  Revision received October 2, 2015  Accepted October 9, 2015  Available online 16 October 2015

http://dx.doi.org/10.1016/j.molmet.2015.10.001

MOLECULAR METABOLISM 5 (2016) 5e18 Ó 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 5
www.molecularmetabolism.com

which is inactivated [8]. triglyceride and impeded weight loss following the return to chow and expressed relative to the expression of the housekeeping genes feeding [16]. Further studies showed that upon Biosystems. remains the only reported PP1-GTS expressed in murine quantity was measured at 260 nm and purity was assessed by the adipocytes. To human adipocytes. indicating with random primers using the Reverse Transcription System (Applied that adipocytes are capable of storing high levels of glycogen. itated in ice-cold 66% (vol. Germany). for mobilization during times of pension. also infiltrating adipose tissue macrophages (ATMs). and glycogen was precipitated by loaded adipocytes exhibit increased autophagic flux. MD) were grown in sus- primarily in liver and skeletal muscle. RGL). There is a paucity of research on the potential physio(patho)logical role of glycogen metabolism in adipose tissue. Radioactivity in impacts their endocrine secretory function. Glycogen syn- glycogen deposition by overexpression of PTG in macrophages pro. Cell monolayers were scraped in 30% (wt/vol. Glycogen content determination demonstrate that glycogen accumulation in adipocytes and macro. which has been linked extract glycogen. Homogenates were take and stimulates glycogen synthesis in adipocytes. adipose tissue-derived macrophages were isolated from the stromal- dinated dephosphorylation of the key glycogenic enzyme glycogen vascular fraction as previously described [20]. respectively. This is an open access article under the CC BY-NC-ND license (http://creativecommons. and 5% CO2. Glycogen synthesis rate obesity redirects glucose metabolic flux into glycogen synthesis in Cells were incubated with 20 mM [U-14C]glucose (0. under the control of the CMV promoter [10]. microfluidic cards) for studies with human samples (See caloric excess-induced expansion of adipose tissue mass. participate in the chronic low-grade inflammation that as previously described [18].Original article 1. were used as cellular models of human subcutaneous and the generation of stress signals and the derangement of carbohydrate visceral pre-adipocytes. cells hallmark of metabolic stress. autophagy and glycogen storage and show that human obesity is associated with glycogen deposition in adipocytes. resulting in Germany). The PP1 catalytic subunit (PP1c) is Cells were infected 7 days after induction of differentiation with an targeted to glycogen particles by glycogen-associated targeting sub. OD260/OD280 ratio.molecularmetabolism. CA). Pp1r3c was Total RNA was extracted from adipose tissue/cells using the RNeasy identified in a two-hybrid screen of a 3T3eL1 adipocyte library [14] Lipid Tissue Midi Kit (Qiagen Science. milligram of protein. System using TaqManÒ Gene Expression Assays (Applied Biosystems) pose tissue was increased. Here we show that hypoxia. Wabitsch (University of Ulm. Ger- accumulation in other organs. In humans. although adipocyte function appeared to be maintained uated by Real-time PCR (qPCR) on a 7900HT Fast Real-Time PCR in these animals. enforced dried papers was counted in a beta-radiation counter./vol. cyclophilin 1A (PPIA) (Hs 04194521_s1) and 18S (Hs 03928985_g1). Homogenates phages contributes to adipose tissue inflammation. As controls. provided by Dr. Foster City. and results were expressed as the percentage of Studies with human clinical samples confirm the interplay between stimulation over basal (control ¼ 100) [10]. glucose can also be stored as glycogen.) KOH. and glycogen was precip- the metabolic alterations in obesity. synthase (GS) by the serine/threonine phosphatase PP1.3) following published protocols [19]. thesis rate was calculated as nanomoles of glucose incorporated per motes polarization towards the M1 pro-inflammatory phenotype. PP1-GTS exhibit tissue and species-specific expression infection. studies in murine models have 2. THP-1 cells (a human monocytic cell line. cell monolayers were scraped into 100 ml of 30% (w/ to obesity-related adipose tissue dysfunction. which directly immersing the paper in ice-cold 66% (v/v) ethanol. were cultured in a standard incubator (21% O2 and 5% CO2). 2. For hypoxia ex- deposition is decreased under pathologic conditions associated with periments.) ethanol. Overall. Hilden. among others [1]. One day after and PPP1R3E. including PPP1R3A (GM. adenovirus expressing murine PTG (Ad-PTG) [21] or GFP (Ad-GFP) units (PP1-GTS).0/). METHODS Adipose tissue is a highly complex metabolic organ with essential roles 2. the Supplementary Experimental Procedures for all evaluated genes). PPP1R3B (GL). hASCs were isolated from the adipose tissue of lean patients energy deficit. or protein targeting to glycogen). PPP1R3D (PPP1R6) carried out for 2 h at a multiplicity of infection (moi) of 50. and might underlie were spotted onto Whatman 3 MM paper.2. The papers were incubated in 6 MOLECULAR METABOLISM 5 (2016) 5e18 Ó 2015 The Authors. v) KOH and homogenates were boiled for 15 min. but not adiponectin. culture medium was depleted of insulin and FBS and and play critical roles in the hormonal regulation of cellular glycogen metabolic experiments were performed 24 h later. Published by Elsevier GmbH. lymphoblast-like cell line. among other Rockville. Excess lipid storage in adipose tissue many) and Lisa-2 cells. elevated levels of glycogen in this model inhibited the mobilization of Results were calculated using the comparative Ct method (2-DDCt). and the associated hyperleptinemia was for in vitro experiments and TaqManÒ Low Density Arrays (Applied independent of fat mass [15]. not only adipocytes but as described [17]. Total RNA and. Möller (University of Ulm. metabolism [9e11]. MD) were induced to differentiate to macrophages with PMA immune cells. We hypothesized that 2. ATCC. 93% N2. ATCC. to date. increases glucose up. Adenoviral transduction lase (GP). Glycogen. For migration While triglyceride stored in adipose tissue is considered the principal experiments.4. provided by Dr. Byosistems. hepatic and skeletal muscle glycogen (BMI 22. such as insulin resistance and type 2 diabetes [3e7]. In this setting.5.1.org/licenses/by-nc-nd/4. In vitro cell cultures in handling and storing excess nutrients and preventing ectopic lipid The SGBS cell line. our data 2. Furthermore.5  0. fully differentiated cells were cultured in a modular incu- obesity. and is a bator flushed with 2% O2. which results in its activation. www. Transgenic overexpression of PTG in adipose tissue in. leads to changes in its metabolic and endocrine functions.3. Human Glycogen synthesis is dynamically regulated by insulin through coor.13]. Although present in low quantities. and the glycogenolytic enzyme glycogen phosphory. Adenoviral infection was PPP1R3C (PTG. protein content in adi. and were induced to differentiate metabolism. spotted onto Whatman 3 MM paper. monocytic THP-1 cells and Jurkat cells (human T cell energy reserve in mammals.05 mCi/mmol).com . was used as a cellular model of human myoblasts. leptin. Quantitative gene expression was eval- Interestingly. Gene expression analysis shown that adipose tissue can store glycogen [12. The human myogenic cell line LHCN-M2 occurs in the adipose tissue of obese individuals [2]. One microgram of RNA was reverse transcribed creases glucose flux into the glycogen synthesis pathway. INTRODUCTION 2. Rockville.

10. This is an open access article under the CC BY-NC-ND license (http://creativecommons. 1. and immunofluorescence confounding and interacting variables was performed using stepwise detection of glycogen was performed as described above.03). mass spectrometry (MS) metabolic profiling of aqueous Quantity One software (Bio-Rad. Germany) and images were nonparametric paired-samples test (Wilcoxon test). tween groups were compared using ANOVA with post hoc Scheffe Microscopy was performed with a Leica DM 4000B fluorescence mi.4 mol/l acetate buffer. tissue samples. ments (Figure S2). values were analyzed by nonparametrical tests. Glucose patients had fasted overnight before collection of blood and adipose release was measured with a glucose kit from Biosystems SA (Bar. Similar to the findings in other cellular models of obesity- mounted on Formvar-coated copper grids.29%  0.5% paraformaldehyde. Published by Elsevier GmbH. serum and plasma. and protein labeled [U-13C]-glucose or natural abundance glucose. MO) for 120 min at 37  C. freshly isolated fat depots teractions with factors were assessed by Pearson’s correlation were fixed in 10% formalin and embedded in paraffin. IL). Despite the differences in the pool size of these metab- University Hospital Virgen de la Victoria (Málaga. Immunofluorescence detection of glycogen independent experiments performed at least in duplicate. cells were incubated overnight at 4  C with expressed as mean value  SD. Statistical Adipocytes grown on coverslips were fixed with 4% (w/v) para.8. with 0. It has been recently shown that hypoxia might the same resin and polymerized at 60  C for 48 h. Immunoreactive bands were visualized with 1. succinate. (PA-SH) method [22]. For in vitro data.06 vs. Samples were adipose tissue inflammation and metabolism in obesity [24e26]. Hypoxia modulates extracellular glucose destination and phosphate buffer (pH 7. To determine 2. and per.2) for 3e6 h at 4  C. and for variables with no Gaussian a monoclonal mouse anti-glycogen antibody in PBS containing 1% distribution values are expressed as median (interquartile range). Nuclear mag- concentration was determined with the BCA Protein Assay Kit (Pierce netic resonance (NMR) spectroscopy of lipid extracts from adipocytes Biotechnology. Statistical analyses negative control. Also. Appropriate Institutional Review Board [U-13C]-glucose did not differ between normoxia and hypoxia treat- approval and biobank informed consent was obtained from all par. Louis. St. ANOVA followed by the protected least-significant different test. Biobank samples included total and fractionated adipose human adipocytes appeared not to be related to lipid biosynthesis or MOLECULAR METABOLISM 5 (2016) 5e18 Ó 2015 The Authors.6. MO) for 10 min at 30  C as a 2. For tween factors as well as the effects of covariates and covariate in- glycogen staining in human adipose tissue. Coverslips were washed with PBS and incubated for 1 h at analysis of expression variables that do not have a Gaussian distri- room temperature with 1:100 Alexa Fluor 568 conjugated goat anti. Plasma and serum samples were stored at 80  C celona. CA). pH 4. experimental results are presented as mean  SEM from 3 to 4 2. RESULTS Small human subcutaneous adipose tissue fragments (3 mm2) were fixed with 2. Hypoxia: and blocked [23]. All dase (SigmaeAldrich. multiple linear regression analysis. Immunoblot analysis whether glucose metabolism was altered by hypoxia. Louis. were all significantly reduced in hypoxia-cultured cells partments at the University Hospital Joan XXIII (Tarragona. the glycolysis end 2. Study selection and sample processing product pyruvate. tissue from subcutaneous and visceral origin. we found that glucose uptake is 2% uranyl acetate in water and lead citrate. correction. expression of glucose transporters GLUT1 and GLUT4 and a non- significant increase in GLUT3 expression (Figure 1B). Spain) in accordance olites. Eugene OR). Statistical analysis was performed with the Statistical Package for the toxylin for histological examination. This examined and photographed using a JEM-1010 electron microscope increase in glucose uptake correlates with significantly greater mRNA (Jeol.org/licenses/by-nc-nd/4. and washed in water for 3e12 h and dehydrated in acetone. Ultrathin sections influence glucose metabolism in adipocytes to prevent oxidative were obtained using a Leica (MZ6) Ultracut UCT ultramicrotome and damage [27]. rehydrated with 2% (v/v) fish skin gelatin. images were captured using the VersaDoc imaging system and However. bution.com . Correction for (5 mm) were deparaffinized and rehydrated. the percentage of [13C]-labeled metabolites arising from with the Declaration of Helsinki. Wetzlar.molecularmetabolism.20%  0. Potential differences between depots were analyzed by croscope (Leica Microsystems. and the tricarboxylic acid (TCA) cycle intermediate Subjects were recruited by the Endocrinology and Surgery De.7. Glycogen was also visualized in duplicate paraffin. MA). Japan). Subsequently. Hercules.0/). sections were increased in SGBS adipocytes subjected to hypoxia (Figure 1A). 7 www.1 M 3..833 mg/ml a-amyloglucosi. Differences in mouse followed by mounting with Pro Long Gold Antifade Reagent clinical variables. 2. Boston. For meabilized with 0. significance was tested with unpaired Student’s t test or one-way formaldehyde. For goat serum. version 15 (SPSS. prior to performing measurements. laboratory parameters.6-diamidino-2-phenylindole.2% Triton X-100 prior to incubation with 5% (v/v) clinical and anthropometrical variables. or expression variables be- with 40 . Tissue sections analysis and General Linear Model Univariate Analysis. Chicago.8. See Supplementary Experimental embedded tissue sections by the periodic acid/salicyloyl hydrazide Procedures for further details. DAPI (Invitrogen Inc. transferred to Immobilon membranes [13C] into lipid structures (Normoxia: 1. extracts from adipocytes subjected to hypoxia or normoxia unexpect- edly revealed that intracellular levels of glucose. Then. Spain).1.5% glutaraldehyde in 0. we measured Cells and human biopsies were lysed and homogenized in RIPA buffer [13C]-enrichment in human adipocytes incubated with uniformly containing Protease Inhibitor Cocktail (SigmaeAldrich). Spain) and (Figure 1C). Other sections were stained with standard hema. no significant differences were detected in the SuperSignal West Femto chemiluminescent substrate (Pierce) and relative total lipid content between the two conditions (Figure S1). Social Sciences software. Half of the samples were incubated with alpha- amylase (SigmaeAldrich. Samples were then promotes glycogen accumulation in human adipocytes washed for 3e12 h with the same buffer and postfixed with 1% Hypoxia has been proposed as an important stimulus for aberrant osmium tetroxide in the same buffer at 4  C for 1 h. Given that high glucose uptake in hypoxia-cultured ticipants.0. embedded in adipocyte dysfunction. St. Sections were stained with associated insulin resistance [28e31]. Interactions be- captured with a Leica DFC 300 FX camera (Leica Microsystems). normal distributed data are goat serum. Equal amounts of total protein were cultured in hypoxia and normoxia revealed a similar incorporation of separated on SDS-PAGE gels.9. Transmission electron microscopy 3. infiltrated with provides a good model system to study mechanisms of obesity-linked Epon (Epon 812) resin for 2 days at room temperature.

www. We also observed a decrease in glycogen nocytochemistry with a glycogen antibody (Figure 1F). Analysis deposition into glycogen (Figure 1D). expressing cells.01 vs Nx. we first analyzed 8 MOLECULAR METABOLISM 5 (2016) 5e18 Ó 2015 The Authors.org/licenses/by-nc-nd/4.05 vs. *p < 0. To determine whether glycogen accumulation influ- related stress conditions. phosphorylase (GP) protein expression in Ad-PTG expressing cells intracellular levels of glycogen significantly decreased in SGBS and (Figure 2C). enforced expression of PTG increases glycogen Lisa-2 adipocytes following 24 h of glucose deprivation. adiponectin [15]. This is an open access article under the CC BY-NC-ND license (http://creativecommons. Accordingly. analysis of glycogen of glycogen levels (Figure 2A) and immunofluorescence staining with content using the a-amyloglucosidase method revealed significant an antibody to glycogen (Figure 2B) demonstrated substantial glycogen increases in intracellular glycogen in hypoxic SGBS and Lisa-2 adi. deposition in PTG-expressing SGBS adipocytes but not in control (Ad- pocytes (Figure 1E). These data show that adipocytes can dynamically regulate glycogen exerts specific effects on the expression of leptin but not their glycogen content in response to glucose availability and obesity. Furthermore. *p < 0. Results are mean  SEM of 3 independent experiments performed in duplicate and are expressed as relative expression to normoxic cells. *p < 0.01 vs. Data from murine stimulation with glucose and insulin increased glycogen accumulation models reveal that enhancement of adipocyte glucose flux into (Figure S3). (D) Glycogen synthesis rate was analyzed in cells incubated with 20 mM [U-14C] glucose. as demonstrated by immu. Nx. Glycogen synthase protein levels were increased in Ad-PTG hypoxia-cultured mature adipocytes obtained by differentiation of hu. (B) GLUT1. (E) Glycogen content in Lisa-2 and SGBS adipocytes incubated in Nx or Hx for 24 h. (P-GS-Ser641) (Figure 2C). *p < 0. Nuclei were stained with DAPI (blue). Published by Elsevier GmbH.01 vs Nx.2. enced the endocrine function of human adipocytes. SGBS adipocytes were placed in hypoxia (Hx: 2% oxygen) or normoxia (Nx: 21% oxygen) for 24 h. Thus.com . We therefore evaluated the impact of function via an autophagy-dependent mechanism hypoxia on glycogen synthesis in adipocytes by measuring the rate of To examine whether glycogen accumulation might lead to metabolic incorporation of radioactively labeled glucose into glycogen after a changes in human adipocytes. pyruvate and succinate levels were analyzed by MS. which correlated with a reduction in its inactive form man adipose-derived stem cells (hASCs). (C) Intracellular glucose. Results are mean  SEM of 3 independent experiments performed in triplicate. Statistical significance was tested with unpaired Student’s t test or one-way ANOVA followed by the protected least-significance different test. Hypoxia enhanced extracellular glucose targeting subunit PTG in SGBS cells by adenoviral infection. Nx. *p < 0. Results are mean  SEM of 3 independent experiments performed in duplicate and are expressed as the percentage of stimulation over control cells. Glycogen deposition in adipocytes modifies their endocrine redirected to glycogen synthesis.05 vs. Human mature adipocytes were incubated in Nx or Hx for 24 h. Results are mean  SEM of 3 independent experiments performed in triplicate and are expressed as the percentage of metabolite levels over control cells. GLUT3 and GLUT4 mRNA expression analyzed by qPCR. whereas synthesis and might inhibit glycogen degradation.0/).molecularmetabolism. (F) Immunofluorescent staining for glycogen in adipocytes obtained by differentiation of hASCs from lean subjects. Nx.Original article A B C * Nx Hx 250 * 3 150 Relative gene expression 200 Glucose uptake 2 100 (% over Nx) (% over Nx) [metabolite] 150 * 100 * 50 * 1 * 50 0 0 0 Nx Hx GLUT1 GLUT3 GLUT4 Nx glc pyr succ Hx D E F SGBS Lisa 250 400 * * Glycogen accumulation 200 * Glycogen synthesis 300 (% over Nx) (% over Nx) 150 200 Glycogen 100 DAPI 100 Nx Hx 50 0 0 Nx Hx Nx Hx Figure 1: Hypoxia increases glucose uptake and promotes glycogen accumulation in human adipocytes. we overexpressed the glycogen- period of glucose deprivation. Increased glycogen content was also observed in GFP) cells. TCA activity. (A) Basal glucose uptake measured during the last 10 min of incubation by quantification of 2-deoxyglucose incorporation. we hypothesized that glucose metabolism might be 3.

0 Ins . (n ¼ 3).0 0 100 0 0 0 0.0 60000 50000 2 * THP-1 migrated cells Jurkat migrated cells 0.5 150 30 1.05.01 vs.05 vs. + E GFP PTG GFP PTG 2. Published by Elsevier GmbH. control cells.0 40000 2. GFP.molecularmetabolism. Quantification of 3 experiments is shown. MOLECULAR METABOLISM 5 (2016) 5e18 Ó 2015 The Authors.com . (D) Gene expression analysis of adipokines and inflammatory markers (n ¼ 4).) 8 GFP PTG 6 4 * 2 * 0 Control ISO CL316 Figure 2: PTG overexpression in human adipocytes increases glycogen content and modulates adipocyte metabolism. *p < 0.01 vs. *p < 0.05 vs. (G) Left Panel -Transduced cells were stimulated with 100 nM insulin for 15 min and total cell extracts were subjected to immunoblotting with antibodies against phosphorylated IRS1 (Tyr612).5 (arbitrary units) 20000 2 10000 10000 1.org/licenses/by-nc-nd/4. Statistical significance was tested with unpaired Student’s t test or one-way ANOVA followed by the protected least significance different test. 9 www. + Ins .5 GFP PTG H 0 0.0 0.0 P-p70 * * * 0. GFP and. *p < 0.0 * (arbitrary units) 4 F 1.PTG or control Ad-GFP and analyzed 48 h post-infection. GFP cells.and Ad-PTG-transduced cells in response to lipolytic stimulus (10 nM Isoproterenol and CL-316243). (E) Leptin.0 0 0 1 GFP PTG 0.0 6 2.5 GFP PTG GFP PTG GFP PTG GFP PTG * Akt phosphorylation 2. *p < 0. GFP. + . GFP (green) and nuclei (blue). A B C Control GFP PTG 150 P-GS (μg glucose/mg protein) * Glycogen content GS total 100 GP DAPI GFP Glycogen GFP 50 PTG 0 GAPDH GFP PTG GFP PTG D G Adipocytes Myocytes GFP PTG 2. (B) Immunofluorescent staining for glycogen (red).u.5 1. PTG. Differentiated SGBS adipocytes were transduced with Ad.0 30000 30000 3 * * p70 phosphorylation 20000 1. Right panel. GFP cells. GAPDH was used as a loading control. Akt (Ser473) and p70S6K (Thr421/Ser424). + 10 # GFP PTG GFP PTG *** Glycerol release (a.0 * * (arbitrary units) * * Leptin levels (pg/ml) APN levels (pg/ml) IL 1b levels (fg/ml) * 2 IL 6 levels (pg/ml) 400 1.0 2. GFP.5 300 * 1. *p < 0.or Ad-PTG-transduced SGBS adipocytes. (C) Equal amounts of total cell extracts were subjected to immunoblotting with antibodies against total and phosphorylated GS (Ser641).5 50000 40000 * 0 0. + . ***p < 0.0 LEP APN IL1b TNF MCP-1 IL6 Ins .5 3 IRS1 phosphorylation 200 40 500 2. (H) Glycerol release was measured in Ad-GFP.001 vs.5 GAPDH 0. (F) Migration of THP-1 and Jurkat cells in response to conditioned medium from Ad-GFP or Ad-PTG-transduced SGBS adipocytes. (A) Glycogen content.001 vs. and were stimulated with 100 nM insulin for 15 min. IL-6. GAPDH was used as a loading control. #p < 0. This is an open access article under the CC BY-NC-ND license (http://creativecommons. *p < 0.0/).LHCNM2 myocytes were cultured for 24 h in conditioned medium from Ad-GFP.5 200 50 10 0.05 vs. Results are mean  SEM of 3 independent experiments performed in duplicate and are expressed as mg glucose/mg protein. Total cell extracts were subjected to immunoblotting with antibodies mentioned above. GP. + .5 0.0 1 100 20 1.5 P-IRS1 Relative gene expression 2. IL-1beta and adiponectin (APN) levels in the medium were analyzed by ELISA. control cells. + Ins .0 * * * P-Akt 1. + .5 1.

Consistent with the increase in leptin expression. an increase in the expression of the active form of mTOR and phosphorylation of its in the sensitivity to the lipolytic agents isoproterenol and CL-316243 downstream targets. To test whether these changes in endocrine proteins including GABARAPL1 and L3CBII (Figure 3A). control cells. increasing evidence from diverse model systems sug- adiponectin showed a similar pattern of gene expression and protein gests a dynamic regulatory cross-talk between autophagy and secretion (Figure 2E). Differentiated SGBS adipocytes (n ¼ 4e5) were transduced with Ad-PTG or control Ad-GFP and analyzed 48 h post-infection. Among these genes. Total cell extracts were subjected to immunoblotting with antibodies against phosphorylated ULK1 (Ser758).org/licenses/by-nc-nd/4.0 2.0 1. the metabolic link proposed previously between lipid and carbohydrate Compared with control cells.5 1. GAPDH was used as a loading control.0/). 10 MOLECULAR METABOLISM 5 (2016) 5e18 Ó 2015 The Authors. (D) Transduced SGBS adipocytes were treated or not with 5 mM 3-MA. Statistical significance was tested with unpaired Student’s t test followed by the protected least-significance different test.5 DAPI 0. L3CBII and PTG. Unexpectedly. only leptin.com . we next assessed however. TNF-alpha increased significantly upon PTG overexpression.5 0. (C) Adipocytes were treated or not with 5 mM MHY1485. MCP-1 and IL-6 gene expression decreased significantly glycogen accumulation alters the endocrine function of adipocytes. Basal adipocyte metabolism was unaffected by glycogen cells (Figure 3A). (E) Immuno- fluorescence detection of glycogen (red).0 Glycogen 0. metabolic gene in glycogen-loaded adipocytes was associated with an inhibition of expression and lactate release were observed in glycogen-loaded AMPK and mTORC1 signaling.5 * 2. glycogen metabolism [33]. forced glycogen deposition in conditioned medium from PTG-overexpressing cells (adip-CM) Ad-PTG-expressing human adipocytes led to an increase in autophagic enhanced the migratory capacity of human monocytic THP-1 cells and flux as revealed by the increased expression of key autophagy-related Jurkat T cells (Figure 2F).32]. Akt and which has been directly related to autophagic degradation of glycogen p70S6K was significantly blunted in Ad-PTG-transduced adipocytes particles with aberrant branching (termed glycophagy) [34. were observed between Ad-PTG cells and Ad-GFP cells in the insulin signaling in glycogen-loaded adipocytes. GAPDH was used as a loading control. IL-1beta and this context.molecularmetabolism. *p < 0.0 0. Published by Elsevier GmbH. Although no differences were detected in A B C D GABARAPL1 P-AMPK P-ULK1 GABARAPL1 LC3BII P-ACC LC3BII PTG STBD1 P-mTOR PTG GAPDH GRP78 P-p70S6K GFP PTG GFP PTG GAPDH CHOP P-ULK1 -3MA +3MA GFP PTG GFP PTG IKB-α P-TSC2 -MHY +MHY IKB-β P-PRAS40 GAPDH PTG GFP PTG GAPDH GFP PTG E F 3. Insulin-induced expression of starch-binding domain-containing protein 1 (STBD1). whereas We next sought to explore the potential mechanism(s) by which adiponectin. www.01 vs. relative to equivalent Ad-GFP cells (Figure 2G). GAPDH was used as a loading control. No differences. Consequently. respectively. (AeB) Total cell extracts from transduced differentiated SGBS cells were subjected to immunoblotting with the indicated antibodies. In (Figure 2D). p70S6K and ULK1.0 GFP PTG 2. IL1-beta and metabolism [16. function might also modulate insulin sensitivity.Original article adipokine and cytokine gene expression in Ad-PTG transduced cells. Intriguingly. Nevertheless. ER stress markers (GRP78. CHOP) and NF-kB signaling obtained in human myocytes cultured for 24 h with adip-CM components (IkBa and IkBb) were unchanged in PTG overexpressing (Figure 2G). Nuclei were stained with DAPI (blue). we detected a decrease adipocytes and control cells (Figure S4). A similar result was Moreover. This is an open access article under the CC BY-NC-ND license (http://creativecommons.35].5 1.0 -3MA +3MA -3MA +3MA -3MA +3MA Figure 3: Enforced glycogen deposition in human adipocytes stimulates leptin secretion through an autophagy-dependent mechanism.0 1. (F) Leptin expression and secretion was analyzed by RT-qPCR and ELISA. however. in Ad-PTG adipocytes relative was evident in glycogen-loaded adipocytes (Figure 2H). mRNA expression of leptin. Total cell extracts were subjected to immunoblotting with antibodies against GABARAPL1 and PTG. phosphorylation of insulin receptor substrate-1 (IRS-1). supporting to Ad-GFP cells (Figure 3B). this apparent activation of autophagy accumulation since similar cellular respiratory rates.5 * Leptin relative levels Leptin mRNA levels * 2.

5 0.05 vs. (n ¼ 3) *p < 0.PTG or control Ad-GFP macrophages. Statistical significance was tested with unpaired Student’s t test or one-way ANOVA followed by the protected least-significance different test. (n ¼ 3) *p < 0. (C) Total cell extracts from transduced differentiated THP-1 cells were subjected to immunoblotting with the indicated antibodies.5 3 3 * GFP Normalized O2 flow per cells 2. Unactivated mac- rophages (M0) were transduced with Ad-PTG or control Ad-GFP and analyzed 48 h post-infection. GAPDH was used as a loading control.05. Nx. *p < 0.01.GFP. Published by Elsevier GmbH.0 0 0 GFP PTG GFP PTG 0. GFP. MOLECULAR METABOLISM 5 (2016) 5e18 Ó 2015 The Authors. (I) Gene expression of representative TCA cycle genes in infected macrophages.0/). (n ¼ 3) *p < 0. (J) High-resolution respirometry studies were performed using an Oroboros-2kTM respirometer. Leak respiration (uncoupled). (B) Immunofluorescent staining for glycogen (red) and GFP (green) in THP-1 macrophages. Results are mean  SEM of 3 independent experiments performed in triplicate. (E) THP-1 and Jurkat cell migration using conditioned media of Ad. (n ¼ 3) *p < 0. ETS capacity (maximal) and ROX (non mitocondrial) values were measured. (n ¼ 3) *p < 0.01.com . (G) Lactate and (H) Succinate accumulation in the conditioned medium of infected THP-1 macrophages.5 1 1 * 1. 11 www.05.molecularmetabolism. Nuclei were stained with DAPI (blue). (D) Expression of representative M1 and M2 marker genes in M0 infected macrophages.0 Routine LEAK ETS ROX Figure 4: Enforced glycogen deposition in THP-1 macrophages stimulates a pro-inflammatory gene expression and glycolytic metabolic profile.01. Routine (basal). This is an open access article under the CC BY-NC-ND license (http://creativecommons.05 vs. (F) Metabolic gene expression profile of THP-1 macrophages infected with Ad-PTG or Ad.org/licenses/by-nc-nd/4. (n ¼ 4) *p < 0. (A) Glycogen synthesis. A B C Control GFP PTG P-GS * Glycogen synthesis GS total (n%over Nx) GP GFP DAPI GFP Glycogen PTG GAPDH Nx Hx GFP PTG D E 6000 8000 3 GFP * Relative gene expression * THP-1 migrated cells Jurkat migrated cells PTG 6000 * 4000 2 4000 * 1 2000 * 2000 0 * 0 0 IL1b IL6 IL12b TNF MCP1 Arg1 IL10 MRC1 GFP PTG GFP PTG M1 Markers M2 Markers F G H 400 150 GFP 3 Relative gene expression * Succinate (μM) PTG 300 Lactate (mM) * 100 * 2 200 1 50 100 * 0 HK2 PFKM PDK4 CPT1a CPT1b ACAA1 SLC25A20 0 0 GFP PTG GFP PTG Glycolysis β-oxidation I J 2.0 gene expression gene expression OGDH relative * SDHB relative 2 2 PTG 1.

Since macrophages can adopt different acti- induced autophagy is dependent on mTORC1 inhibition.06 5.39  14. although similar cellular respira- tissue and promote macrophage M1 polarization tory rates were observed. suggesting that the mitochondrial: non mitochondrial tissue from PTG KO mice and wild-type (WT) littermates [32]. Moreover.46  0. Also. M0). This is an open access article under the CC BY-NC-ND license (http://creativecommons. contributing to adipose tissue inflammation and systemic secretion [41] (Figure 4H). we analyzed several glycogen- (human cells) with whole adipose tissue (murine model).63 5. a decrease in residual oxygen consumption To study the in vivo function of PTG in adipose tissue inflammation.94 6.org/licenses/by-nc-nd/4.3. SBP: Systolic Blood Pressure.87  11.35  0.5a DBP (mmHg) 68.19  0. Notably.22  25.19  1. hypoxia enhanced extracellular glucose obese subjects (Figure 5A).91 BMI (kg/m2) 23. these results highlight metabolic gene expression profile.59 29.80  19.75  2. glycogen-loaded macrophages showed higher succinate dehydroge- nase (SDHB) and alpha-ketoglutarate dehydrogenase (OGDH) gene 3. supporting the accumulating evi. expression (Figure 4C).48 3. which glycogen levels in human adipocytes might modify their endocrine has recently been associated with enhanced glycolysis and IL-1b functions. a indicate significant differences between the means of the different groups: a: p < 0. Taken together.66 1.29  14.001: b: p < 0. Table 1 e Anthropometric and biochemical variables in the two cohorts studied for mRNA and protein expression levels. We therefore (Figure 4B).90  10.4-alpha) branching enzyme 1 (GBE1) and PP1- man THP-1 macrophages.02a 23. Published by Elsevier GmbH.0/).26a Glucose (mmol/L) 4. We found a significant reduction in the glycogen accumulation on the metabolic state and phenotype of hu. but not lymphocyte migration was increased with conditioned medium (CM) glycogen accumulation (Figure 3E). correlating with enhanced GS activity and decreased GP used the mTOR activator MHY1485 [36] to question whether glycogen.29  1.58 1.56  0. we of leptin secretion in human adipocytes and suggest that modulation of detected an increase in the TCA cycle intermediate succinate.97  11.35 1.10  21.63 Cholesterol (mmol/L) 5. and consistent with pub- inhibited its secretion (Figure 3F). suggesting that mTORC1 regulation increased intracellular glycogen levels in THP-1 macrophages might be dependent on PRAS40 regulation (Figure 3B).84  16.56a HOMA-IR 1. Incubation of human adipocytes with 3-MA blocked glycogen-loaded M0 cells (Figure 4D). BMI: body mass index.77  0.21 1.11  1. we found that the commitment of glycogen-loaded dence that autophagy serves as a new mechanism to control protein M0 macrophages to the M1 phenotype was also evident in the trafficking and secretion [38].74  1. Glycogen levels control the pro-inflammatory state of adipose expression (Figure 4I).4. respiration ratio might be increased in Ad-PTG M0 macrophages Consistent with the link suggested between PTG overexpression and (Figure 4J).69 Insulin (mIU/ml) 4.07  3.21 42.67 15.62 79. which was directed towards the glycogen-autophagy intersection as a novel regulatory mechanism glycolysis (Figure 4F) and lactate release (Figure 4G). the pro-inflammatory state (Figure 2).53 1.33  0.Original article TSC2 phosphorylation at the Thr1462 residue.11  18. Differences in the expression of some with autophagy gene expression in human obesity of these genes (mainly MCP-1 and IL-6) between human cellular and To determine whether obesity and insulin resistance altered glycogen murine models could be a consequence of comparing adipocytes metabolism in human adipose tissue.15  6. lished reports [40]. 12 MOLECULAR METABOLISM 5 (2016) 5e18 Ó 2015 The Authors. insulin resistance. 3-MA treatment had no from PTG-overexpressing macrophages compared with CM from effect on the PTG-induced increase in leptin gene expression but control cells (Figure 4E).33 42.95 5. Consistent with an increase in TCA activity.com .57a Waist (cm) 83.33a 83.44 6.77 135.52  3. we in mTOR-induced ULK1 phosphorylation at Ser758 detected in the assessed whether enforced glycogen deposition influenced macro- presence of MHY1485 was associated with a decrease in L3CBII phage polarization. Altered expression of glycogen metabolic genes correlates relative to WT mice (Figure S5). PTG KO mice display reduced expression of several inflammatory gene markers in adipose tissue 3.32  1.4  11. mirroring expression levels correlated negatively with body mass index (BMI).98 SBP (mmHg) 121.15  0.36 Triglycerides (mmol/L) 1.71  4.11  0.14  12.70 41.84 1.68  15. First.90 76.47  9. Interestingly. The increase vation states (classical M1 or alternative M2 activation) [39].47  11. we observed a decrease our observations in adipocytes (Figure 2BeC). we confirmed that.18 5. we further explored the effects of summarized in Table 1). DBP: Diastolic Blood Pressure.01 HDLc (mmol/L) 1.77b 78. More importantly.45  0.10  0.58  13. Finally.40  0.12  1. We analyzed M1 and M2 gene expression markers expression (Figure 3C). HOMA-IR: homeostasis model assessment of insulin resistance index. in Ad-PTG M0 (unactivated) macrophages and control cells (Ad-GFP phagy in the regulation of leptin production by glycogen accumulation.94 131. we (ROX) was detected in Ad-PTG macrophages compared with Ad-GFP first analyzed the inflammatory gene expression profile in adipose macrophages. Because related metabolic genes (Supplementary Experimental Procedures) in inflammatory responses in adipose tissue are dynamically regulated a well-characterized human cohort (clinical and laboratory data through innate immune cells.90b 110.68 5.52  0. www. to examine a potential role for auto.39 1.molecularmetabolism. monocyte and the PTG-induced expression of GABARAPL1 (Figure 3D). Bivariate analysis revealed that GBE1 gene deposition in macrophages into glycogen (Figure 4A).9 99. First cohort Second cohort Lean Obese Lean Obese N 19 37 10 38 Sex (male/female) 13/6 22/15 5/5 16/22 Age (years) 51.96 58.11  0.49a 4.17  0.01 (Student t test). expression of glucan (1.29 1. We observed an increase in the classical M1 markers IL-1b and we utilized 3-methyladenine (3-MA) as a pharmacological inhibitor of MCP-1 in parallel with a decrease in the M2 markers Arg1 and IL-10 in autophagy [37].78 116.7  7. PTG overexpression in P-PRAS40 levels (Thr246). similar to human GTS 3E (PPP1R3E) mRNA in the subcutaneous adipose tissue (SAT) of adipocytes (Figure 1D).83b The results are given as the mean  SD. Furthermore.81  2.26  0.

(D) Bivariate correlations between glycogen metabolic genes (GBE1 and PPP1R3E) and ULK1. Of note. PPP1R3E mRNA expression in SAT and VAT depots from the first cohort (n ¼ 56).034 1. Interestingly.5 0.0 Lean r=-0.0 * 0. lean.0 0.001 ULK1 relative 1. Given the relationship found between glycogen mRNA expression (Figure 5D).001) and PPP1R3E (B ¼ 0.265 r=-0.com . G and H).262 r=-0. *p < 0. To date.0 0. (A) GBE1 and PPP1R3E mRNA expression in SAT depots of subjects from the first cohort (n ¼ 56) divided into two groups according to BMI (lean: BMI<25 and obese: BMI>25).0/). (F) PPP1R3C and PPP1R3E mRNA expression in SAT and VAT depots compared to skeletal muscle (SM) (n ¼ 9) *p < 0.5 0 2 4 GBE1 relative gene PPP1R3E relative 0.org/licenses/by-nc-nd/4.001) Experimental Procedures).4 * 0. Potential differences between depots were analyzed by nonparametric paired-samples test (Wilcoxon test).5 4 4 GBE1 relative gene PPP1R3E relative 1.0 0. *p < 0. 13 www.0 1.0 1. P < 0. we found that (Figure 5C). Published by Elsevier GmbH. PPP1R3C. Data are presented as median with interquartile range (A and C) and as mean  SEM (E. Data comparisons were made by Kruskal Wallis test for multiple groups and Mann Whitney test for two groups.5 * 5 1 1 * * * 0 0 0 0.0 0.5 0.509 1.01. A B 2.2 0.5 0.0 1.0 expression gene expression 0. More importantly.5 0.0 GABARAPL1 ULK1 3B 3C 3D 3E F G H SAT VAT 20 4 4 1.0 1.5 1 1 0.0 2 2 * 0. *p < 0.molecularmetabolism.649. PPP1R3D. P ¼ 0.0 SM SAT VAT AD SV AD SV AD SV Figure 5: Human obesity is associated with changes in adipose glycogen metabolism and autophagy gene expression.001 VAT gene expression ULK1 relative p<0. Additionally.035 p=0.5 1. whereas PPPR13E was negatively associated with waist circumference between glycogen metabolic genes (GBE1 and PPP1R3E) and ULK1 and weight (Figure 5B).6 1.0 0 0 0. we analyzed the gene expression analysis adjusting for age and gender revealed that ULK1 predicts profile of autophagy-related genes in the same cohort (Supplementary GBE1 (B ¼ 0. multiple regression metabolism and autophagy in vitro.548.0 r=0.037 p=0.5 0.264 Relative gene expression Obese p=0.5 1. gene expression in SAT. (C) GABARAPL1 and ULK1 mRNA expression in SAT depots of subjects from the first cohort.0 Lean Relative gene expression 2. (E) PPP1R3B. Interactions between factors as well as the effects of covariates and covariate interactions with factors were assessed by Pearson’s correlation analysis and General Linear Model Univariate Analysis. consistent positive correlations were found PPP1R3E was the most predominant PP1-GTS isoform in human VAT MOLECULAR METABOLISM 5 (2016) 5e18 Ó 2015 The Authors. F.0 * 10 2 2 0.5 gene expression PPP1R3E relative gene expression expression * 3 3 1.5 Relative gene expression Relative gene expression 3C 3C Relative gene expression SAT GBE1 relative gene * 3E 3E 3 3 VAT 15 expression 1. PTG (PPP1R3C) is the only reported dividuals exhibited lower mRNA levels of GABARAPL1 and ULK1 PP1-GTS expressed in murine adipocytes (15).05. This is an open access article under the CC BY-NC-ND license (http://creativecommons.5 SAT Relative gene expression gene expression p<0.0 20 30 40 50 100 150 50 100 150 GBE1 PPP1R3E BMI waist (cm) weight (kg) C D E 1.8 Obese 2.01 vs.778 r=0. (B) Correlation between SAT GBE1 and PPP1R3E mRNA expression and clinical variables. SAT depots from obese in. (G) PPP1R3C and PPP1R3E and (H) GBE1 mRNA expression in isolated adipocytes (AD) and stromal vascular fraction (SV) from SAT and VAT (n ¼ 6.01. paired adipose biopsies).

Published by Elsevier GmbH.769 p= 0.0 0 Lean Ob Lean Ob + amylase E F Lean Obese Lean Obese CTRL Alexa568 H I Glycogen content (μg/mg protein) 30 r= 0.0 8 3 (vs lean) expression 1. our in vivo with greater levels of GS protein in SAT (Figure 6A).10 vs. n ¼ 5 and obese. (I) Bivariate correlations between glycogen content in mature adipocytes and BMI and Leptin mRNA expression (Pearson’s correlation analysis). *p < 0. Statistical significance was tested with unpaired Student’s t test or one-way ANOVA followed by the protected least significance different test. GAPDH was used as a loading control. GAPDH was used as a loading control.98 pg/ml).5 * 10 * (vs Ob low IR) GS Relative protein 2.028 Glycogen content (μg/mg protein) 20 G 10 A B C D BMI Lean 0 Glycogen content (μg/mg protein) Lean Ob r= 0. (G) Glycogen detection in SAT from lean and obese subjects by transmission electron microscopy (TEM).Original article and SAT depots (Figure 5E).0 20X 40X 20X 40X -2. n ¼ 7). lean subjects. insulin sensitivity. (F) Representative glycogen immunofluorescent detection in SAT biopsies from lean and obese individuals.5 * * -1.49  7. Quantification is shown (n ¼ 4). high IR). (C) Representative immunoblotting of GBE1 protein expression in isolated adipocytes (AD) and stromal vascular fraction (SV) from SAT depots of lean and obese patients. we confirmed that PPP1R3E mRNA is agreement with mRNA levels. Quantification is shown (n ¼ 4).5 0. In demonstrated [23].0 -0. with a nificant differences in mRNA expression. we performed protein expression HOMA-IR values (>3.com . PPPR13E mRNA is highly cohort (clinical and laboratory data summarized in Table 1). where a high level of this isoform was previously than in lean individuals (58. PPP1R3E and GS protein expression in SAT from subjects of the second cohort allocated into two groups according to BMI (lean: BMI<25. No 3. GAPDH was used as a loading control.5.0 1 * 0. indicating specificity of staining for glycogen.96  10. obese subjects presented a significant found predominantly in mature adipocyte cells in human adipose tissue reduction in GBE1 and PPP1R3E protein expression in the SAT depot and not in other cells from the stromal-vascular (SV) pool (Figure 5G). obese low IR subjects.5 2 0. (A) Representative immunoblotting of GBE1. Although we found no sig- Similarly.0 0. (E) Glycogen immunofluorescence (red) on macrophages extracted from lean or obese human adipose tissue. Black arrows indicate macrophage-like structures. however. *p < 0. 19.2). in which expressed in adipose tissue (VAT and SAT) relative to human skeletal circulating leptin levels were confirmed to be higher in obese subjects muscle (Figure 5F).2 and high insulin resistance (high IR): HOMA-IR>3. *p < 0.0/). Human obesity is associated with enhanced protein levels of significant differences were found in GBE1 protein expression between GS and glycogen stores in adipose tissue both groups (low IR vs. (D) Representative histological analysis of SAT from lean and obese subjects using periodic acid-Schiff (PAS) staining for glycogen. obesity was also associated lower signal in the SV fraction (Figure 5H). As a whole.003 E F G H Obese Leptin relative gene expression Figure 6: Human obesity leads to glycogen deposition in adipocytes.05 vs. Pretreatment with a-amylase eliminated PAS- positive staining in tissue sections. www.5 0. obese patients with high To validate our gene expression data. The negative control with the secondary antibody is also shown.2) presented a significant decrease in PPP1R3E studies in a selected number of subjects from a second independent protein expression in SAT depots compared with patients with low A B C D SV Lean Ob Lean Obese Lean Ob Ob low IR Ob high IR GS GBE1 GAPDH 10X PPP1R3E Adipocytes Lean Ob GS GS GAPDH GAPDH * 20X 5 SV 14 Adipocytes Relative protein expression Relative protein expression * 12 GBE1 4 PPP1R3E 2. (H) Glycogen content in mature adipocytes isolated from SAT of lean (n ¼ 9) and obese (n ¼ 10) subjects using a-amyloglucosidase method. PPP1R3E and GS protein expression in SAT of obese subjects (Ob) classified according to insulin resistance assessed by HOMA-IR (low insulin resistance (low IR): HOMA-IR<3.608 * p= 0. 14 MOLECULAR METABOLISM 5 (2016) 5e18 Ó 2015 The Authors. compared with lean subjects (Figure 6A). To explore a observations consolidate a significant association between glycogen potential link between glycogen metabolism in adipose tissue and metabolism and autophagy in human adipocytes. Nuclei were stained with DAPI (blue). lean subjects.0 40X 6 1.molecularmetabolism.0 1. Representative images are shown (n ¼ 6).5 1. obese subjects from the second cohort were clas- sified according to their insulin resistance assessed by HOMA-IR. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. ATMs (black arrows).05 vs. GBE1 is essentially restricted to mature adipocytes. (B) Representative immunoblotting of GBE1. Ob: BMI>25. Indeed. Quantification is shown in the lower panels. In addition.5 -1.05 vs. Glycogen is visualized as small dark granules (black arrows).

org/licenses/by-nc-nd/4. DISCUSSION which glycogen accumulation controls mTORC1 activity. where glycogen has been revealed as an metabolism.43]. sensing kinase mTOR. Periodic acid-Schiff (PAS) glycogen as an inflammatory metabolic signal in adipose tissue. our results establish that observed suggesting that mTORC1 regulation in our model might be adipose tissue glycogen deposition may underlie whole-body meta. Further. autophagy flux in glycogen- microscopy (TEM) of fat tissue from biopsy specimens demonstrated loaded adipocytes correlates with a negative regulation of the nutrient- that glycogen could be detected as small dark granules in the cyto. it is thought that in adipose tissue. inhibition of method in mature adipocytes isolated from SAT depots of lean and mTORC1 is sufficient to induce autophagy in the presence of nutrients obese subjects. also observed in human adipose tissue with implications for obesity. Critically. glycogen content in mature adipocytes positively corre- dendritic cells [52e55]. but a decrease in P-PRAS40 revealed a positive association between glycogen content and BMI and (Thr246). which correlates with a secretion pattern to one that is characterized by a decrease in the anti. the presence of a glycogen sensor in the mTOR pathway has also been Glucose is one of the major energy substrates for adipose tissue recently suggested in liver. and we provide evidence that glycogen accumulation results context behave as if they are in a nutrient and energy deprivation state in a damaging metabolic and secretory profile in these cells. hyper. Furthermore. lates with BMI and leptin expression.59]. our data establish Our study provides insights into the metabolic consequences of PPP1R3E as the major PP1-GST isoform expressed in human adipo- glycogen overcapacity in human adipose tissue.47] or Intriguingly. an upstream negative regulator of mTORC1 [61]. obese patients with the highest tion.com . the proposed networking of glycogen with autophagy is endoplasmic reticulum stress [48]. Accordingly.2). reflective of an M1 methodological approaches to assess whether human obesity could be phenotype as previously described [41]. In fact. Interestingly. which. the potential role of AMPK inactivation. which bolsters emerging discoveries linking auto- lean and obese subjects (Figure 6E). AMPKb subunit has been revealed as a regulatory domain that inhibits pose tissue dynamics has received little attention. we quantified glycogen content using the a-amyloglucosidase detected in other cellular systems [58. supporting the link found in vitro inflammatory cytokines [54]. PRAS40 phosphorylation could be AKT-dependent or eindependent [62]. such as hypoxia (this study) [45]. We suggests a disturbance in some cellular energetic sensor and point to postulate that the increase of basal glucose uptake observed in mTORC1 signaling as a key regulator of adipocyte metabolism in obesity-linked conditions. Consequently. rection of glucose metabolism to glycogenesis. However. decrease in GBE1 and PPP1R3E. Surprisingly. we observed higher rates of lipolysis in (Figure 6B). Unexpectedly. which has been previously associated with plasm of subcutaneous adipocytes from obese patients (Figure 6G). Collectively. was also leptin expression (Figure 6I). enforced glycogen deposition modifies the adipocyte crease in glycogen deposition in adipocytes. pathological conditions. might contribute to the insulin resistant state asso- suggesting that alterations in GS might be related more to adiposity ciated with obesity. Additionally. it is lead to a myriad of local effects. insulinemia [28]. Published by Elsevier GmbH. Leptin is a potent chemoattractant for monocytes. and also activates the expression of other pro.values (HOMA-IR<3. Here we not unreasonable to suggest a model where adipocytes may sense demonstrate that a redirection of glucose flux to glycogen synthesis pathological accumulation of glycogen through an mTORC1 mecha- occurs as a response to hypoxia in human adipocytes and macro. glycolysis and succinate release. Obese subjects display an obvious in- [49e51]. The reduced levels of some terms of insulin sensitivity and immune cell recruitment were detected glycogen metabolic genes detected in obese adipose tissue may MOLECULAR METABOLISM 5 (2016) 5e18 Ó 2015 The Authors. 15 www. including hypoxia [44]. Remarkably. glycogen accumulation has a pro- than to insulin resistance status. and additional studies are needed to elucidate the mechanism by 4. might be associated with a redi. but more importantly with an increase inflammatory adipokine adiponectin and an increase in leptin secre. PRAS40-dependent. Similar to accumulation provokes damaging effects as previously demonstrated PTG. where glucose can be converted to cross-talk is considered an important regulator of autophagy [63]. staining of glycogen in SAT depots revealed an increase in total cellular Of note. in agreement with previous findings in PTG-transgenic mice [15]. bivariate analysis ferences in TSC2 phosphorylation. transmission electron Generally activated by nutrient deprivation. neutrophils and Remarkably. AMPK phosphorylation by upstream kinases [64] and Ulk1 might also Adipose tissue expansion in response to positive energy balance can directly phosphorylates and inactivates AMPK [65]. PPP1R3E overexpression results in glycogen accumulation in in several cell types including neurons and retinal pigment epithelium SGBS adipocytes (Figure S7). glycogen-loaded adipocytes did not display dif- in obese mature adipocytes (Figure 6H). we found no differences in GS protein glycogen-loaded adipocytes. both in adipocytes and ATMs (Figure 6D). pro-inflammatory cytokine activation [46. combined with their altered expression in lean subjects stratified as low/high IR (Figure S6). Un. This is an open access article under the CC BY-NC-ND license (http://creativecommons. and functional integrity of this organ in terms of glucose important coordinator of glucose and lipid metabolism [32]. of GS protein expression. autophagy crosstalk mechanism controlling leptin secretion in hu- Immunofluorescent staining of glycogen in isolated macrophages from man adipocytes. Together. Nevertheless.0/). secretory profile. and contrary to murine models [15]. It is also noteworthy that although AMPK-mTOR-Ulk1/2 metabolic like skeletal muscle and liver.molecularmetabolism. our data identifies related to changes in fat glycogen storage. the glycogen-binding domain on the glycogen metabolism in human adipose tissue and its effect on adi. as Finally. manner. we report for the first time a new regulatory glycogen- glycogen in obese subjects. (Figure 6F). bolic alterations found in human obesity. Importantly. nism independent of AMPK. Thus. degree of insulin resistance present the highest protein levels of GS. Moreover. The fact that adipocytes from an obese phages. Indeed. as well as in SAT depots phagy to biosynthetic processes [32] and cytokine release [56]. we found significantly greater GS in PTG-expressing adipocytes. Contrary to the in vivo data from aP2- protein expression in the SAT depot of those insulin-resistant subjects PTG transgenic mice [16]. and PTG (PPP1R3C) is mainly found in the SV fraction. deleterious effects in between adipocyte glycogen and leptin. but not with ER stress or NFKb signaling. autophagy activation [57]. we glycogen (glycogenesis). enforced glycogen GS protein expression was detected both in adipocytes and SV cells deposition in inactive macrophages increases pro-inflammatory gene from SAT of obese subjects (Figure 6C). glucose is found that the accumulation of glycogen in human adipocytes and used mainly to provide energy or to supply glycerol phosphate for subsequently autophagy activation is associated with mTORC1 but also triacylglycerol synthesis [43]. A significant increase of glycogen levels was detected [60]. confirmed these findings. Adipocyte glycogen cytes. produced in a TSC2-independent. uptake is crucial for regulating intermediate metabolism [42. we used different expression. the significant increase in inflammatory effect in macrophages. Accordingly.

V. AGF. glycogen synthesis and autophagy supports the link between both processes.1016/j.org/licenses/by-nc-nd/4. which might underlie the patho. PMG-R and ARS contributed to discussion [1] Krahmer. Glycogen-loaded adipocytes present an increased autophagic flux. KR. www. APPENDIX A. XD. Additionally. CS.molecularmetabolism. None. and wrote the manuscript. L ¼ lipid droplet. Otto Baba for his generous gift of the monoclonal population (Figure 7). adipocytes and ATMs exhibit a redirection in glucose metabolism to glycogen synthesis (glycogenesis).. JV and SFV conceived the study. The Spanish Biomedical Research Center in Diabetes and Associated Metabolic described in obese and diabetic patients [66e69]. this study provides the first evidence of adipose tissue III. our study suggests metabolic disturbance in adipocytes as a potential primary event in obesity-related inflammation that should be The authors have declared that no conflict of interest exists. In the context of human obesity. glycogen deposits in inactivated (M0) macrophages promotes M1 polarization. ULK1 was revealed as one of the main determinants ACKNOWLEDGMENTS of GBE1 and PPP1R3E mRNA expression in SAT depots and was downregulated in obese subjects.doi. Disorders (CIBERDEM) (CB07708/0012) is an initiative of the Instituto de Salud Carlos In conclusion. program (CP10/00438 and CP13/00188. T. a shift from glycolytic metabolism to glycogenesis (ERDF). CNR and PP carried out the experiments and generated data.. respectively) from the Fondo de Inves- ́ genesis of obesity and insulin resistance. CONFLICT OF INTEREST theless.001. Pau Gama for their help function of adipocytes by a mechanism dependent on autophagy flux with respirometry experiments and Dr. suggesting that adipose tissue glycogen might be a metabolic inflammatory signal in human obesity. directly impacts their secretory function.. Published by Elsevier GmbH. had full access to all the data in the study against the pathological accumulation of glycogen in adipocytes.. cussed data. Maria Vinaixa and Sara Samino from Metabolomics Platform of occurs in adipose tissue. We postulate that in an tigacion Sanitaria (FIS) co-financed by the European Regional Development Fund obesity setting. considered in the development of new therapeutic strategies to alle- viate obesity-associated metabolic disorders. We also thank Dr. Thus.com . Whether glycogen mishandling is a primary glycogen antibody. SF-V and LG-S acknowledge support from the “Miguel Servet” tenure-track glycogen as an inflammatory signal. N ¼ nucleus. in turn. which. On and take responsibility for the integrity of the data and the accuracy of the other hand. JV and SFV are the guarantors 16 MOLECULAR METABOLISM 5 (2016) 5e18 Ó 2015 The Authors.Original article Figure 7: Adipose tissue glycogen accumulation as a potential trigger of obesity-linked inflammation in humans. dis- droplets and human disease. We thank Dr. GS ¼ glycogen synthase. ES. 2013. as well as an increase in the M1 polarized macrophage the manuscript.10. the correlation observed between several markers of the data analysis. G ¼ glycogen accumulation. Farese Jr. This is an open access article under the CC BY-NC-ND license (http://creativecommons. EMBO Molecular Medicine 5:905e915. encouraging immune cell recruitment and insulin resistance. MMG.2015. R. molmet. which directly determines the secretory CIBERDEM for their help with metabolomic analysis data. LGS and FT carried out part of the study selection and human sample processing. OY and MAR carried out the metabolomics REFERENCES studies. Kenneth McCreath for helpful comments on activation.0/). represent the onset of a counter regulatory mechanism as a defense of this work and.C. as such. Balancing the fat: lipid and reviewed the manuscript. VCM. SUPPLEMENTARY DATA AUTHOR CONTRIBUTION Supplementary data related to this article can be found at http://dx. We hypothesize that the reduction in This study was supported by grants from the Spanish Ministry of Economy and GABARAPL1 and ULK1 gene expression detected in our cohorts might Competitiveness (SAF2012-36186 to SF-V. disturbance or an indirect consequence of obesity is difficult to establish at the present time and awaits further investigation. N. Walther. ME.org/10. PI14/00228 to JV and PI12/02355 to represent a compensatory mechanism for autophagy induction FJT).

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