GLP-1 Receptor Signaling in Astrocytes Regulates Fatty Acid Oxidation, Mitochondrial Integrity, and Function
Authors: Christian Heilinger
Aleksandra Trifunovic
Katharina Timper
Luis Varela
Jens C Brüning
Almudena Del Río-Martín
Heiko Backes
Patrick Giavalisco
Anna Lena Cremer
Stephan Bremser
Jens Alber
Peter Kloppenburg
Tamas L Horvath
Hendrik Nolte
Journal: Cell Metabolism (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: FGF-21; GLP-1; astrocytes; energy homeostasis; glucose metabolism
Affiliations: Max Planck Institute for Metabolism Research, Department of Neuronal Control of Metabolism, Gleueler Str. 50, 50931 Cologne, Germany; Center for Endocrinology, Diabetes and Preventive Medicine (CEDP), University Hospital Cologne, Kerpener Str. 26, 50924 Cologne, Germany; Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD) and Center of Molecular Medicine Cologne (CMMC), University of Cologne, Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany., Max Planck Institute for Metabolism Research, Department of Neuronal Control of Metabolism, Gleueler Str. 50, 50931 Cologne, Germany., Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD) and Center of Molecular Medicine Cologne (CMMC), University of Cologne, Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany; Institute for Zoology, Biocenter, University of Cologne, Zuelpicher Str. 47B, 50674 Cologne, Germany., Max Planck Institute for Biology of Aging, Joseph-Stelzmann-Str. 9b, 50931 Cologne, Germany., Program in Integrative Cell Signaling and Neurobiology of Metabolism, Department of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06520, USA., Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD) and Center of Molecular Medicine Cologne (CMMC), University of Cologne, Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany., Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD) and Center of Molecular Medicine Cologne (CMMC), University of Cologne, Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany; Institute for Mitochondrial Diseases and Aging, Medical Faculty, University of Cologne, 50931 Cologne, Germany., Max Planck Institute for Metabolism Research, Department of Neuronal Control of Metabolism, Gleueler Str. 50, 50931 Cologne, Germany; Program in Integrative Cell Signaling and Neurobiology of Metabolism, Department of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06520, USA; Department of Anatomy and Histology, University of Veterinary Medicine, 1078 Budapest, Hungary., Max Planck Institute for Metabolism Research, Department of Neuronal Control of Metabolism, Gleueler Str. 50, 50931 Cologne, Germany; Center for Endocrinology, Diabetes and Preventive Medicine (CEDP), University Hospital Cologne, Kerpener Str. 26, 50924 Cologne, Germany; Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases (CECAD) and Center of Molecular Medicine Cologne (CMMC), University of Cologne, Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany; National Center for Diabetes Research (DZD), Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany. Electronic address: .

Abstract:  Astrocytes represent central regulators of brain glucose metabolism and neuronal function. They have recently been shown to adapt their function in response to alterations in nutritional state through responding to the energy state-sensing hormones leptin and insulin. Here, we demonstrate that glucagon-like peptide (GLP)-1 inhibits glucose uptake and promotes β-oxidation in cultured astrocytes. Conversely, postnatal GLP-1 receptor (GLP-1R) deletion in glial fibrillary acidic protein (GFAP)-expressing astrocytes impairs astrocyte mitochondrial integrity and activates an integrated stress response with enhanced fibroblast growth factor (FGF)21 production and increased brain glucose uptake. Accordingly, central neutralization of FGF21 or astrocyte-specific FGF21 inactivation abrogates the improvements in glucose tolerance and learning in mice lacking GLP-1R expression in astrocytes. Collectively, these experiments reveal a role for astrocyte GLP-1R signaling in maintaining mitochondrial integrity, and lack of GLP-1R signaling mounts an adaptive stress response resulting in an improvement of systemic glucose homeostasis and memory formation. 

Retracted: No

PubPeer comments:
 Just wondering if the authors could comment on the clamp data which look really interesting. Why are the EGP values negative when measured during the clamp? Does this indicate switching from liver glucose production to uptake? Also, does the large difference in clamp EGP between genotypes indicate the GLP1 mouse has increased hepatic glucose uptake? This could support the liver glycogen data. , 
 How is this considered a well-done clamp or a clamp experiment at all? Why is no glucose infused for 30 min or so? Who starts the GIR at 80 mg kg min and then drops it? Most seriously, there is no plausible reason to have a EGP (or Ra) or -40ish. ![file] ![file] , 
 We thank the commenters for their interest in this study. The commenters are perfectly right that physiologically a negative value for suppression of HGP is neither expected nor plausible. Although, negative HGP values during euglycemic-hyperinsulinemic clamps have been described in the literature, particularly at high glucose infusion rates as observed in our study, this relates to insufficient modeling. The clamp studies in the present study had been performed years ago, early in the project. Since then we have in the meantime optimized our clamp protocol according to the guidelines by Mouse Metabolic Phenotyping Centers, Vanderbilt-NIDDK (begin baseline primed-continuous tracer infusion at least 90 minutes prior to clamp start [3-3H]glucose is added to the glucose infusate to minimize deviations in blood glucose specific activity during the clamp no priming insulin dose when using an insulin infusion of 4mU*kg-1*min-1 and performing the clamp for 2h immediate start of low dose glucose infusion with begin of insulin infusion). This technique has proven successful in subsequent studies to prevent large negative measurements of endogenous glucose production. Nevertheless, since the clamps in this study had been performed according to an old protocol also with a delayed onset of the glucose infusion, we have clearly stated in the results section of the manuscript that the results of negative HGP values cannot be interpreted. Given that the focus of the study was not on the detailed assessment of tissue-specific changes in glucose metabolism, we had decided to include the clamp data only as a further indicator of increased systemic insulin sensitivity in mice lacking the GLP-1R in astrocytes. Katharina Timper, Jens Bru00fcning , 
 Not an isolated issue. https: pubpeer.com publications 6540D4B87F4BFE85C71202B70912F0 https: pubpeer.com publications 371E5713E415618E965F7752F239B9 , 
 By the way there are numerous issues that can result in a negative EGP and these were dealt with decades ago - see the work of Rizzau2019s group, Bergman and Finegood as well as Hother-Nielsen and Beck-Nielsen. So the knowledge is out there and has been for a long time. , 
 #5 ...indeed. Certainly not a new issue. The fact that it was included, statistics were performed, it passed through review, and was published is troublesome. As noted by prior comments, negative Ra values occur and have been discussed for decades. That only strengthens the argument that -40 is absurd. , 
 a bigger issue than clamping is that: 1) Gfap is not restricted to astrocytes. Many cell types in peripheral tissues express Gfap including Schwann cells, perivascular cells, etc.. 2) the distribution of glp1r in the brain is well known and does not include astrocytes (PMID: 29095968; PMID: 9886047; PMID: 25380238). , 
 Also note regarding clamp data that basal rates seem to be ~ 5 mg kg min. Very low for a mouse. , 
 We would like to respond to the comments posted by Bartsia Peruviana stating on one hand the problem of GFAP-Cre mice deleting in peripheral tissues and stating on the other hand the absence of GLP-1R expression in astrocytes. The commenter is right that GFAP-Cre mice also delete in peripheral cell types. Thus, this comment holds true for all 100 studies having employed these mice for studying astrocytes. Nevertheless, to further substantiate that GFAP-expressing cells in the CNS, specifically in the hypothalamus contribute to the observed phenotype upon deletion of the GLP-1R employing GFAPCreERT2 mice, we have also specifically reduced GLP-1R-expression in GFAP-expressing cells of this brain region by injecting an AAV expressing Cre under control of the GFAP promoter into the hypothalamus of GLP-1Rflox flox mice. These data are provided in Suppl. Fig. 8 of our manuscript. They revealed that this intervention phenocopied the improvement in glucose tolerance and changes in hypothalamic glucose uptake as assessed via PET scan and as observed in GLP1Rflox floxGFAPCreERT2 mice. Regarding the expression of GLP-1R in astrocytes we have very carefully studied it through multiple complementary approaches in our present study. First, we detect GLP-1R mRNA expression in GFAP-positive cells via RNAscope based in situ hybridization and abrogation of this signal in whole body GLP-1R knockout mice (Suppl. Fig. 1). Second, we reveal uptake of fluorescently labeled Liraglutide - a GLP-1R agonist - in GFAP positive cells of control but not of GLP-1R knockout mice (Suppl. Fig. 1). Third, we reveal GLP1-R mRNA and protein expression in cultured primary hypothalamic astrocytes and its reduction upon specific inactivation (Suppl. Fig. 3). Furthermore, we have paid careful attention to the functional validation of altered Liraglutide uptake to GFAP-positive astrocytes of GLP1Rflox floxGFAPCreT2 mice (Fig. 3). Collectively, we have carefully assessed GLP-1R expression in GFAP-expressing cells and its alteration in our experimental models. These data are in line with previously published work revealing GLP-1R expression in GFAP-expressing astrocytes again through complementary approaches as well as functional effects of GLP-1 or GLP-1R agonists on astrocytes: 1.tAlvarez E et al., J Neurochem, 1996: mRNA in situ hybridization detected GLP-1R mRNA expression in glial cells in rat brain (Fig. 3) 2.tChowen JA et al., Neuropeptides, 1999: Increased GLP-1R mRNA expression in GFAP-expressing astrocytes in rat brain surrounding mechanical lesions (Fig. 1) 3.tYun SP et al., Nature Medicine, 2018: mRNA and protein expression of GLP-1R in cultured primary mouse, GFAP-positive astrocytes (Fig. 3A-C) 4.tReiner DJ et al., Journal of Neuroscience, 2016: colocalization and internalization of fluorescent-labeled Exendin-4 in NTS GFAP-expressing astrocytes in rats (Movie 1); prevention of uptake of fluorescent-labeled Exendin-4 in NTS GFAP-expressing astrocytes by icv injection with the GLP-1R antagonist Exendin 9-39 in rats (Movie 2; Fig. 2-4) 5.tShan Y et al., Journal of Neuroinflammation, 2019: Immunohistochemistry detection of GLP-1R in GFAP astrocytes in rat brain (Fig. 5A), protein expression of GLP-1R in cultured primary mouse, GFAP-positive astrocytes via Western blot (Fig. 2A, B) 6.tIwai T et al., Neuroscience Research, 2006: Binding of fluorescent-labeled GLP-1 to GFAP-positive cultured rat astrocytes, detection of GLP-1R mRNA in cultured, primary rat astrocytes (Fig. 1). 7.tBao Y et al., Cellular Physiology and Biochemistry, 2015: mRNA and protein expression of GLP-1R in cultured primary cortical rat astrocytes and specific reduction via siRNA-mediated knockdown (Fig. 3A, B). 1.tReiner DJ et al., Journal of Neuroscience, 2016: Exendin-4 treatment increases [Ca2+] signaling in rat NTS astrocytes which was reduced upon pretreatment with Exendin 9-39 (Fig. 5); GLP-1, Exendin-4 and liraglutide increased cAMP in an immortalized rat type-1 astrocyte cell line (DI-TNC1) (Fig. 6). Intra-NTS injection of Exendin-4 reduced chow-food-intake, which was attenuated upon fluorocitrate injection in rats (Fig. 8). 2.tMarina N et al., Glia, 2017: The GLP-1 analog Exendin-4 elicits [Ca2+]i responses in cultured brainstem astrocytes transduced to express GCaMP6f, Exendin-4-induced [Ca2+]i responses were abolished in the presence of the GLP-1R antagonist Exendin 9-39 (Fig. 4B, D) 3.tShan Y et al., Journal of Neuroinflammation, 2019: Exendin-4 reduces expression of VEGF and cytokines in primary mouse astrocytes in the setting of experimental stroke 4.tIwai T et al., Neuroscience Research, 2006: GLP-1 prevents LPS-induced IL-1b production in cultured rat astrocytes In contrast, the studies cited by Bartsia Peruviana (PMID: 29095968; PMID: 9886047; PMID: 25380238), although representing elegant work investigating the distribution of GLP-1R immunoreactivity and mRNA expression in rodent and non-human primate brain, to our knowledge have not specifically addressed the question of GLP-1R co-expression in GFAP-positive cells. Katharina Timper, Almudena del Rio Martin, Jens Bru00fcning for the authors , 
 Thank you for the answer. The GFAP stains in supplementary figure 1 are very muddled and it is not really possible to tell at such low magnification if the glp1r mRNA or liraglut binding occurred in the soma of astrocytes. , 
 I in part agree with #7 and #10 that the main evidence for Glp1r mRNA in vivo (Fig. S1A-B or even S10A) seems rather dubious. This is quite an important point as the whole story is built on this evidence. For many of the alleged positive astrocytes (arrows), Glp1r+ or liraglutide+ dots appear not co-localizing with GFAP at all. And for those few that seem to co-localize, orthogonal views may better reveal whether this is truly the case, rather than the result of either saturation of the GFAP channel or misleading merging of confocal stacks. Presently I dont really see a robust evidence for any Glp1r expression in astrocytes, and any strong conclusion on this based on the few available staining data is quite debatable. For panel S1B, in HIP and NTS acquisitions, each red dot appears somewhat double positive also for GFAP (which itself should show a filamentous pattern, not dotted), raising further concerns on the acquisition parameters utilized for these images and possible signal cross-talk. Similar concerns for evidence for FGF21 mRNA expression in astrocytes in vivo apply (Fig. 5C). , 
 #11 Moreover, at least two thorough anatomical studies using highly-sensitive reporters provided evidence of absence of Glp1r mRNA in astrocytes: 1. Cork, S. C., Richards, J. E., Holt, M. K., Gribble, F. M., Reimann, F., Trapp, S. (2015). Distribution and characterisation of glucagonu2010like peptideu20101 receptor expressing cells in the mouse brain. Mol Metab, 4(10), 718u2013731. 2. A novel mouse model of glucagon-like peptide-1 receptor expression: A look at the brain. Graham DL, Durai HH, Trammell TS, Noble BL, Mortlock DP, Galli A, Stanwood GD. J Comp Neurol. 2020 Oct;528(14):2445-2470. ,