Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2014) 239 – 245 nanomedjournal.com
Imaging liver and brain glycogen metabolism at the nanometer scale Yuhei Takado, MD, PhD a, b,⁎, Graham Knott, PhD c , Bruno M. Humbel, PhD d , Stéphane Escrig, PhD a , Mojgan Masoodi, PhD e, f , Anders Meibom, PhD a, g,⁎, Arnaud Comment, PhD b,⁎ a
Laboratory for Biological Geochemistry, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Institute of Physics of Biological Systems, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland c Centre Interdisciplinaire de Microscopie Electronique, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland d Electron Microscopy Facility, Université de Lausanne, Lausanne, Switzerland e Nestlé Institute of Health Science, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland f Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Canada g Center for Advanced Surface Analysis, Institute of Earth Sciences, University of Lausanne, Lausanne, Switzerland Received 21 May 2014; accepted 12 September 2014 b
Abstract In mammals, glycogen synthesis and degradation are dynamic processes regulating blood and cerebral glucose-levels within a well-defined physiological range. Despite the essential role of glycogen in hepatic and cerebral metabolism, its spatiotemporal distribution at the molecular and cellular level is unclear. By correlating electron microscopy and ultra-high resolution ion microprobe (NanoSIMS) imaging of tissue from fasted mice injected with 13C-labeled glucose, we demonstrate that liver glycogenesis initiates in the hepatocyte perinuclear region before spreading toward the cell membrane. In the mouse brain, we observe that 13C is inhomogeneously incorporated into astrocytic glycogen at a rate ~ 25 times slower than in the liver, in agreement with prior bulk studies. This experiment, using temporally resolved, nanometer-scale imaging of glycogen synthesis and degradation, provides greater insight into glucose metabolism in mammalian organs and shows how this technique can be used to explore biochemical pathways in healthy and diseased states. © 2014 Elsevier Inc. All rights reserved. Key words: Carbon-13; Glucose metabolism; NanoSIMS; Hepatocytes; Astrocytes
Background Animal tissues can store glucose in the form of glycogen, a large polysaccharide (up to about 5⋅10 4 glucose equivalents called glucosyl residues) that is mostly found in muscles and in the liver. 1 To maintain blood glucose level within a narrow physiological range around 5 mM, the liver breaks down glycogen to release glucose into the blood stream. Glycogen is also stored in the mammalian brain, in the cytoplasm of glial The authors declare no competing financial interests. This work was supported by the Swiss National Science Foundation (grant PP00P2_133562 to A.C), the Centre d’Imagerie BioMédicale (CIBM) of the UNIL, UNIGE, HUG, CHUV, EPFL, and the Leenards and Jeantet Foundations. The NanoSIMS instrument was funded in part by an ERC advanced grant 246749 (BIOCARB and by the Ecole Polytechnique Fédérale de Lausanne to AM. ⁎Corresponding authors. E-mail addresses: [email protected] (Y. Takado), [email protected] (A. Meibom), [email protected] (A. Comment). http://dx.doi.org/10.1016/j.nano.2014.09.007 1549-9634/© 2014 Elsevier Inc. All rights reserved.
cells in concentrations approximately 1% of those found in the liver. 2 It is nevertheless the largest energy reserve in the brain and is present at concentrations of around 5 μmol glucosyl residues/g wet weight in humans and rodents. 3 Several approaches have been used to determine the concentration and localization of glycogen in mammalian tissues in order to assess its role in hepatic and cerebral energy metabolism. Isotopic labeling is a particularly appropriate and powerful method for this purpose since it allows the measurement of metabolic rates in vitro and in vivo. Enrichment of small molecules in otherwise low-abundance stable isotopes (e.g. 13C or 15 N) does not affect their biological functions nor does it affect the actions of enzymes involved in their transport and metabolism. One of the most widely used techniques to measure glycogen in vivo is 13C nuclear magnetic resonance (NMR) spectroscopy. 4,5 It can be used in conjunction with the administration of 13C-glucose to non-invasively assess the in vivo time evolution of hepatic and cerebral glycogen metabolism
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in an animal or a human for several hours. 6,7 However, due to its relatively low sensitivity, it is not possible to achieve high spatial resolution (i.e. better than ~ 0.5 cm 3). 8 Glycogen can also be detected indirectly through the NMR signal of water, allowing a better sensitivity than 13C and therefore a sensibly higher spatial resolution. 9 Another technique based on a glucosamine radioisotope has been recently proposed to measure glycogenesis, the formation of glycogen, in vivo in tumors via positron emission tomography (PET). 10 Although noninvasive, these in vivo techniques have a spatial resolution restricted to about 1 mm 3. To achieve higher resolution, tissues can be imaged ex vivo. Radioactively labeled ( 3H and 14C) glucose has been used to study hepatic and cerebral glycogen metabolism, but the spatial resolution of the associated imaging technology (autoradiography) is still limited to about 10 μm. 11,12 Techniques to investigate the formation dynamics of liver and brain glycogen at the subcellular level have not been available until now and the capability to observe the spatiotemporal distribution of glycogen at subcellular length scales would provide a fundamentally new tool in the study of glucose metabolism in both healthy and pathological tissue. Here we demonstrate the capability to obtain time-resolved nanometer-scale images of hepatic and cerebral glycogen formation following the administration of 13C-labeled glucose into awake animals. This is made possible through the combined use of electron microscopy (EM) and ultra-high spatial resolution ion microprobe (NanoSIMS) imaging, which provides maps of isotopic ratios on thin-sections of biological tissue with a spatial resolution of around 100 nm. 13,14 This allows both the precise localization of glucosyl residues within subcellular compartments and quantitative isotopic (here 13C/ 12C) ratio determination, providing the capability to trace exactly where and when in the compartments of single cells new glycogen deposits are formed during the isotopic labeling experiment.
Methods Animal preparation and injection protocol All experiments were conducted in accordance with relevant guidelines and regulations and approved by the local ethics committee (office véterinaire du canton de Vaud). Adult (10-24 week-old) wild-type NMRI male mice were fasted overnight prior to each experiment (n = 8). A 1.1 M aqueous [U- 13C]glucose solution (99% 13C isotopic enrichment, SigmaAldrich, Buchs, Switzerland) was prepared and a series of doses corresponding to 1 mg/g were injected intraperitoneally (i.p.) in awake mice at time intervals of 0, 10, 20, 60, 90, 120 and 150 min. The animals were placed in their cage after each injection. Blood samples were taken before each injection to monitor the time evolution of the glucose plasma level. At different time points (1, 2, and 3 h, respectively), after the initial injection, mice were deeply anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally injected) and transcardially perfused with 2 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 M phosphate buffer at pH 7.4. 15 All procedures involving the animals were performed using humane methods.
Tissue sample preparation for EM and NanoSIMS The brain and liver of each mouse were removed 2 h after perfusion and 70-μm-thick vibratome (Leica VT1200, Leica Biosystems, Nussloch, Germany) sections were cut through the liver and in the coronal plane through the neocortex. The sections were then washed in cacodylate buffer (0.1 M, pH 7.4), postfixed for 40 min in 1.5% potassium ferrocyanide and 1% osmium tetroxide, followed by 40 min in 1% osmium tetroxide alone, and 40 min in 1% uranyl acetate in water. They were then dehydrated through a series of increasing concentrations of ethanol, and infiltrated with Durcupan resin (Fluka, Buchs, Switzerland). The sections were flat embedded between glass slides in fresh resin and left for 24 h at 60 °C for the resin to harden. Thin sections at 70 nm thickness were cut with the diamond knife and collected on formvar coated alphanumeric grids or single slot grids with a pioloform support film. EM imaging Images of brain and liver ultra-thin section were recorded in a transmission electron microscope (Tecnai Spirit, FEI Company, Eindhoven The Netherlands) at 80 keV with a CCD camera (4 k × 4 k Eagle cameria, FEI Company). Individual EM images were collaged together using the Photoshop software (Adobe Systems, San Jose, CA, USA). Images were placed into different layers of a single file and one by one their transparency was increased to view the image below. This layer was then moved manually until its correct alignment was seen. This was repeated for all layers until the final collaged image was complete. Quantitative NanoSIMS isotopic imaging To image and quantify in situ the subcellular distribution of 13Cenriched glycogen within liver and brain cells, the exact same areas imaged by EM were analyzed with a NanoSIMS 50 L ion microprobe (CAMECA SAS, Gennevilliers, France), enabling direct correlation of ultrastructural (EM) and isotopic (NanoSIMS) images. EM grids were mounted on 10-mm diameter aluminum stubs and coated with about 10 nm gold. They were bombarded with a 16 keV primary ion beam of Cs + (1-3 pA) focused to a spot size of about 100-150 nm on the sample surface. Secondary molecular ions 12 14 − C N and 13C 14N − were simultaneously collected in electron multipliers at a mass resolution sufficient to avoid potentially problematic isobaric interference of 12C 15N − on 13C 14N −. Charge compensation was not necessary. Isotopic images ranging in size between 15 × 15 μm and 50 × 50 μm containing 256 × 256 pixels were obtained by rastering the primary beam across the sample surface with a dwell-time of 5 ms. Images were processed using the L’IMAGE® software (Larry R. Nittler, Carnegie Institution of Washington, Washington, DC, USA). 13 12 C/ C ratio distribution maps were obtained by taking the ratio between the drift-corrected 13C 14N − and 12C 14N − images. 13Cenrichments were expressed in the conventional delta notation: δ C ð‰Þ ¼ 13
C mes −1 1000; C nat
where Cmes is the measured 13C/ 12C ratio and Cnat is the average natural 13C/ 12C ratio measured regularly in non-labeled, identically
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Figure 1. Nanometer scale imaging of liver glycogen synthesis from [U- 13C]glucose. (A) EM image of liver thin section prepared from a fasted mouse 60 min (top) and 120 min (bottom) after the first injection of [U- 13C]glucose, respectively; yellow arrowheads point to glycogen granules; (B) Quantitative NanoSIMS 13 C-enrichment map of precisely the same region on the same thin sections; (C) 13C-enrichment profile along the line drawn on the NanoSIMS image in (B).
prepared samples throughout the period of NanoSIMS analyses (0.01104 ± 0.00004 (1 S.D.), n = 12). NanoSIMS isotopic maps were overlaid onto the EM images, using Photoshop software. The individual 12C 14N − images, which define cellular structures, such as the nucleus and mitochondria were initially overlaid to achieve an accurate correspondence between the NanoSIMS and EM field of view. Then individual NanoSIMS 13C/ 12C ratio maps were placed in the same position as their corresponding 12C 14N − images, which were subsequently removed. The 13C/ 12C ratio maps were then made 50% transparent to simultaneously view the underlying EM images. Statistical analysis Statistical analyses were performed by using the OriginPro 9.0G software (OriginLab, Northampton, MA, USA). Kolmogorov– Smirnov first tested the data for normality. Student’s t test was used for comparing groups of data. Results were considered significant at 5%. Throughout the paper, values given are mean ± S.D., and error bars show S.D.
Results The fixation, staining and embedding protocol used in this experiment is a conventional method for preparing tissue for electron microscopy, and clearly shows the cell ultrastructure, including the glycogen granules within both the brain and liver.
These are densely stained particles with a diameter of 10-40 nm, often clustered into larger aggregates (Figure 1, A). The spatial distribution of the 13C/ 12C isotopic ratio was imaged by NanoSIMS over the same area viewed in the EM in brain and liver sections (see e.g. Figure 1, B). Overlays of the NanoSIMS and EM images show local isotopic enrichments associated with the glycogen granules. It also gives the possibility to quantitatively determine the spatial variation of the 13 C enrichment along specific directions within the sections (see Figure 1, C). Spatially-resolved time evolution of glycogen synthesis in hepatocytes Large areas (minimum of 90 × 90 μm 2) of liver tissue from each experiment were imaged with EM, as illustrated in Figure 2, A. The glycogen granules appeared to be homogenously distributed among the hepatocytes identified in the larger collaged EM views. In these images, the localization of individual cell nuclei (highlighted by white lines in Figure 2, A) and plasma membranes (blue lines in Figure 2, A) is easily distinguished, as are glycogen aggregates. Strong heterogeneities in the spatial distribution of the isotopic ratio were observed and clear shifts from the natural 13C/ 12C ratio were detected in all liver sections obtained from mice exposed to [U- 13C] glucose for 60 min or longer (Figures 1, B and 2, A). Divisions between the perinuclear region surrounding the nucleus, and the peripheral region adjacent to the cell membrane (Figure 2, A) showed that the labeling is initially stronger in the
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Figure 2. Mapping the subcellular distribution of glycogen formation. (A) EM (left column) and 13C/ 12C NanoSIMS (right column) images of hepatocytes after 60, 120, and 180 min of exposure to [U- 13C]glucose, revealing the spatiotemporal progression of glycogen formation; white contours outline the hepatocyte nuclei, blue contours outline the hepatocyte membranes, and red contours divide each hepatocyte into a perinulear (i.e. close to nucleus) and a peripheral region; Small red and blue circles outline glycogen occurrences in the perinuclear and peripheral regions of each hepatocyte, respectively; (B) Histogram comparing 13 C-enrichment of liver glycogen located in two different regions of the hepatocytes; The initial (first 120 min) preferential region of enrichment, namely the perinuclear region, is later (180 min) labeled at about the same level as the peripheral region,.
perinuclear region (Figures 1, C and 2, B) in the time series analysis. The isotopic enrichment of glycogen aggregates located within both the perinuclear and peripheral regions of the
hepatocytes was determined from the NanoSIMS analyses and plotted as a function of the duration of the labeling period (Figure 2, B).
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Figure 3. Brain glycogen synthesis from [U- 13C]glucose. (A) EM image of brain thin section prepared from a fasted mouse 180 min after the first injection of [U- 13C]glucose; black arrows point to glycogen granules; (B) Quantitative NanoSIMS 13C-enrichment map correlated with the EM image; (C) 13C-enrichment profile along the line drawn on the NanoSIMS image in (B).
Spatial distribution of
13
C-labeled cerebral glycogen
In EM images of thin sections through the entire cortical thickness, glycogen granules were unambiguously identified (black arrows in Figure 3) and were exclusively found in the cytoplasm of astrocytes. 16 As expected from earlier studies, the density of glycogen aggregates was much lower in brain tissue than in liver tissue. All glycogen aggregates detected in astrocytes exhibit a 13C/ 12C ratio above the natural isotopic ratio. As observed in hepatocytes, the glycogen granules are not homogeneously labeled. However, there was no specific labeling pattern. On average, in animals euthanized after 3 h of labeling, the isotopic enrichment of glycogen was much lower in brain slices than in liver slices, with mean values of δ 13C = 170 ± 140 ‰ compared to δ 13C = 4220 ± 700 ‰, respectively (Figure 4). From these data, it is apparent that the 13C-enrichment observed in brain glycogen after 3 h is about 25 times lower than in the liver. Discussion The experiments presented here demonstrate how it is possible to study the incorporation of glucose into hepatic and cerebral glycogen at high resolution. Although it is known
that tissue glycogen content might decrease due to degradation during the standard preparation steps used in the present work, 17 the carbon isotopic ratio maps will not be affected if such loss affects glycogen granules in a homogeneous fashion. In vivo NMR studies have used similar doses of 13C-labeled glucose to study glycogen metabolism in rodents and it is therefore possible to compare these studies with the results obtained here. A liver glycogen 13C-enrichment of about 11 000 ‰ (i.e. a factor of 12) compared to the natural 13C/ 12C ratio of 0.011 was measured after 3 h in rats that were injected 6 mg/g [1- 13 C]glucose. 7 This is about twice the mean enrichment deduced from our data: 4220 ± 700 ‰ observed in liver glycogen aggregates after 3 h following the injection of 7 mg/g [U- 13C]glucose. However, our study used [U- 13C]glucose instead of [1- 13C]glucose and most labeled glucose-6-phosphate (glucose-6-P) that is incorporated into glycogen is formed from [U- 13C]pyruvate molecules deriving from glycolysis since it has been demonstrated that gluconeogenic compounds are the major carbon source for hepatic glycogen. 18 A substantial fraction of the 13C is thus lost in the form of 13CO2. 19,20 With regard to our NanoSIMS analyses, it must also be considered that 12C ions originating from the embedding material (in particular epoxy resin) introduced during sample preparation will to some extend contribute to a lowering of the apparent isotopic enrichments.
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Figure 4. Incorporation of 13C from [U-13C]glucose into astrocytic and hepatocytic glycogen. (A) Schematic representation of the incorporation of 13C into liver and brain glycogen from blood [U-13C]glucose; (B) Average isotopic enrichment in liver (orange) and brain (blue) glycogen aggregates after 3 h labeling through i.p. injection of [U-13C]glucose. The observed mean value was δ13C = 170 ± 140‰ and δ13C = 4220 ± 700‰ in the brain and the liver, respectively.
Overall, although there is a relatively large standard deviation on the mean enrichment from NanoSIMS analyses of small parts of the liver, the 13C-enrichment we observe is comparable to the enrichment obtained from the in vivo 13C NMR analyses. The fact that the average 13C/ 12C ratio determined with NanoSIMS in brain glycogen after 3 h is about 25 times lower than the ratio measured in liver glycogen is also in good agreement with earlier in vivo 13C NMR studies. Indeed, the glycogen turnover rate of ~0.5 μmol⋅g −1⋅h −1 previously observed in rat brain by 13C NMR21 corresponds to a rate that is about 30 times lower than the 15 μmol⋅g − 1 ⋅h −1 deduced from the rate of net glycogen synthesis and
degradation measured in rat liver. 5 The general consistency with the in vivo 13C NMR observations supports the fact that the NanoSIMS images capture and correctly quantify glycogen formation, even if some of the glycogen might be lost in the sample preparation. Therefore, the proportion of glycogen that might be lost through sample preparation and/or rapid metabolic degradation associated with cell death was similar in brain and liver tissue. In addition, if the 13 C incorporation into glycogen follows a last-in first-out scheme, as expected from earlier studies, 22,23 only a relatively small fraction of glycogen was lost because the relative 13C-enrichment was comparable to that measured in vivo by 13C NMR. If we nevertheless assume that a considerable part of brain glycogen was lost during cell death, we must conclude that the glucosyl residues that were labeled within the 3 h of the experiment are not preferentially the ones used, via glycogenolysis, as emergency energy reserve. Our data demonstrate that initial glycogenesis from 13C-labeled glucose molecules injected into fasted animals is taking place with a strong preference for the perinuclear region of hepatocytes. This observation is in contrast to previous in vitro experiments performed using [ 14C]glucose combined with EM, 24,25 where it was observed that glycogen synthesis starts at the cell periphery, i.e., near the plasma membrane. This discrepancy may simply be a reflection of differences in the way in vitro and in vivo cells handle glucose. In the in vitro studies, it was shown, as expected, that the 14 C-label is highly correlated with glycogen synthase concentration, which was not measured in the present study. The early and strong presence of 13C-labeled glycogen in the perinuclear region observed in our study is itself consistent with the fact that glucokinase is localized in the nucleus of hepatocytes when glucose level is low, before being released into the cytosol as soon as the blood glucose level increases. 26,27 No significant deviation from the natural fractional 13C/ 12C ratio was observed inside the hepatocyte nuclei, and if glycogen was present as suggested in an earlier study, 28 it was around or below the detection limit of the NanoSIMS, which is about 50 ‰, possibly because the turnover rate was much slower than inside the cytosol. Abnormal glycogen metabolism has been associated with disorders affecting the central nervous system. Inability to inhibit glycogen synthesis in neurons is the basis of diseases such as Lafora disease, 29 and impaired activity of glycogen branching enzyme occurs in adult polyglucosan body disease. 30 Furthermore, it is now well established that glycogen turnover is altered in many types of tumor cells. 31 Nanometer scale imaging of glycogen metabolism represents a powerful new tool for investigating glycogenassociated disorders at an entirely new level of resolution. Our results open the way for new types of metabolic imaging that will provide fundamental insights into cellular use of a variety of metabolic substrates in both healthy and diseased mammalian tissue. Acknowledgments We thank Dr. Céline Loussert (Electron Microscope Facility at University of Lausanne) for expert advice and technical assistance.
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Y. Takado et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2014) 239–245 2. Nelson SR, Schulz DW, Passonneau JV, Lowry OH. Control of glycogen levels in brain. J Neurochem 1968;15:1271-9. 3. Brown AM. Brain glycogen re-awakened. J Neurochem 2004;89:537-52. 4. Choi IY, Tkac I, Ugurbil K, Gruetter R. Noninvasive measurements of [1- 13C]glycogen concentrations and metabolism in rat brain in vivo. J Neurochem 1999;73:1300-8. 5. David M, Petit WA, Laughlin MR, Shulman RG, King JE, Barrett EJ. Simultaneous synthesis and degradation of rat liver glycogen. An in vivo nuclear magnetic resonance spectroscopic study. J Clin Invest 1990;86:612-7. 6. Choi IY, Gruetter R. In vivo c-13 nmr assessment of brain glycogen concentration and turnover in the awake rat. Neurochem Int 2003;43:317-22. 7. Shulman GI, Rothman DL, Smith D, Johnson CM, Blair JB, Shulman RG, et al. Mechanism of liver-glycogen repletion in vivo by nuclear magnetic resonance spectroscopy. J Clin Invest 1985;76:1229-36. 8. van Heeswijk RB, Pilloud Y, Morgenthaler FD, Gruetter R. A comparison of in vivo 13C MR brain glycogen quantification at 9.4 and 14.1 T. Magn Reson Med 2012;67:1523-7. 9. van Zijl PCM, Jones CK, Ren J, Malloy CR, Sherry AD. MRI detection of glycogen in vivo by using chemical exchange saturation transfer imaging (glycoCEST). Proc Natl Acad Sci U S A 2007;104:4359-64. 10. Witney TH, Carroll L, Alam IS, Chandrashekran A, Nguyen QD, Sala R, et al. A novel radiotracer to image glycogen metabolism in tumors by positron emission tomography. Cancer Res 2014;74:1319-28. 11. Coimbra A, Leblond CP. Sites of glycogen synthesis in rat liver cells as shown by electron microscope radioautography after administration of glucose-h3. J Cell Biol 1966;30:151-75. 12. Swanson RA, Morton MM, Sagar SM, Sharp FR. Sensory stimulation induces local cerebral glycogenolysis — demonstration by autoradiography. Neuroscience 1992;51:451-61. 13. Hoppe P, Cohen S, Meibom A. Nanosims: technical aspects and applications in cosmochemistry and biological geochemistry. Geostand Geoanal Res 2013;37:111-54. 14. Lechene C, Hillion F, McMahon G, Benson D, Kleinfeld AM, Kampf JP, et al. High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry. J Biol 2006;5:1-30. 15. Knott GW, Holtmaat A, Trachtenberg JT, Svoboda K, Welker E. A protocol for preparing gfp-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis. Nat Protoc 2009;4:1145-56. 16. Cataldo AM, Broadwell RD. Cytochemical identification of cerebral glycogen and glucose-6-phosphatase activity under normal and experimental conditions: I. Neurons and glia. J Electron Microsc Tech 1986;3:413-37. 17. Guth L, Watson PK. A correlated histochemical and quantitative study on cerebral glycogen after brain injury in the rat. Exp Neurol 1968;22:590-602.
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Imaging liver and brain glycogen metabolism at the nanometer scale Yuhei Takado, MD, PhD a, b,⁎, Graham Knott, PhD c , Bruno M. Humbel, PhD d , Stéphane Escrig, PhD a , Mojgan Masoodi, PhD e, f , Anders Meibom, PhD a, g,⁎, Arnaud Comment, PhD b,⁎ a
Laboratory for Biological Geochemistry, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Institute of Physics of Biological Systems, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland c Centre Interdisciplinaire de Microscopie Electronique, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland d Electron Microscopy Facility, Université de Lausanne, Lausanne, Switzerland e Nestlé Institute of Health Science, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland f Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Canada g Center for Advanced Surface Analysis, Institute of Earth Sciences, University of Lausanne, Lausanne, Switzerland Received 21 May 2014; accepted 12 September 2014 b
Abstract In mammals, glycogen synthesis and degradation are dynamic processes regulating blood and cerebral glucose-levels within a well-defined physiological range. Despite the essential role of glycogen in hepatic and cerebral metabolism, its spatiotemporal distribution at the molecular and cellular level is unclear. By correlating electron microscopy and ultra-high resolution ion microprobe (NanoSIMS) imaging of tissue from fasted mice injected with 13C-labeled glucose, we demonstrate that liver glycogenesis initiates in the hepatocyte perinuclear region before spreading toward the cell membrane. In the mouse brain, we observe that 13C is inhomogeneously incorporated into astrocytic glycogen at a rate ~ 25 times slower than in the liver, in agreement with prior bulk studies. This experiment, using temporally resolved, nanometer-scale imaging of glycogen synthesis and degradation, provides greater insight into glucose metabolism in mammalian organs and shows how this technique can be used to explore biochemical pathways in healthy and diseased states. © 2014 Elsevier Inc. All rights reserved. Key words: Carbon-13; Glucose metabolism; NanoSIMS; Hepatocytes; Astrocytes
Background Animal tissues can store glucose in the form of glycogen, a large polysaccharide (up to about 5⋅10 4 glucose equivalents called glucosyl residues) that is mostly found in muscles and in the liver. 1 To maintain blood glucose level within a narrow physiological range around 5 mM, the liver breaks down glycogen to release glucose into the blood stream. Glycogen is also stored in the mammalian brain, in the cytoplasm of glial The authors declare no competing financial interests. This work was supported by the Swiss National Science Foundation (grant PP00P2_133562 to A.C), the Centre d’Imagerie BioMédicale (CIBM) of the UNIL, UNIGE, HUG, CHUV, EPFL, and the Leenards and Jeantet Foundations. The NanoSIMS instrument was funded in part by an ERC advanced grant 246749 (BIOCARB and by the Ecole Polytechnique Fédérale de Lausanne to AM. ⁎Corresponding authors. E-mail addresses: [email protected] (Y. Takado), [email protected] (A. Meibom), [email protected] (A. Comment). http://dx.doi.org/10.1016/j.nano.2014.09.007 1549-9634/© 2014 Elsevier Inc. All rights reserved.
cells in concentrations approximately 1% of those found in the liver. 2 It is nevertheless the largest energy reserve in the brain and is present at concentrations of around 5 μmol glucosyl residues/g wet weight in humans and rodents. 3 Several approaches have been used to determine the concentration and localization of glycogen in mammalian tissues in order to assess its role in hepatic and cerebral energy metabolism. Isotopic labeling is a particularly appropriate and powerful method for this purpose since it allows the measurement of metabolic rates in vitro and in vivo. Enrichment of small molecules in otherwise low-abundance stable isotopes (e.g. 13C or 15 N) does not affect their biological functions nor does it affect the actions of enzymes involved in their transport and metabolism. One of the most widely used techniques to measure glycogen in vivo is 13C nuclear magnetic resonance (NMR) spectroscopy. 4,5 It can be used in conjunction with the administration of 13C-glucose to non-invasively assess the in vivo time evolution of hepatic and cerebral glycogen metabolism
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in an animal or a human for several hours. 6,7 However, due to its relatively low sensitivity, it is not possible to achieve high spatial resolution (i.e. better than ~ 0.5 cm 3). 8 Glycogen can also be detected indirectly through the NMR signal of water, allowing a better sensitivity than 13C and therefore a sensibly higher spatial resolution. 9 Another technique based on a glucosamine radioisotope has been recently proposed to measure glycogenesis, the formation of glycogen, in vivo in tumors via positron emission tomography (PET). 10 Although noninvasive, these in vivo techniques have a spatial resolution restricted to about 1 mm 3. To achieve higher resolution, tissues can be imaged ex vivo. Radioactively labeled ( 3H and 14C) glucose has been used to study hepatic and cerebral glycogen metabolism, but the spatial resolution of the associated imaging technology (autoradiography) is still limited to about 10 μm. 11,12 Techniques to investigate the formation dynamics of liver and brain glycogen at the subcellular level have not been available until now and the capability to observe the spatiotemporal distribution of glycogen at subcellular length scales would provide a fundamentally new tool in the study of glucose metabolism in both healthy and pathological tissue. Here we demonstrate the capability to obtain time-resolved nanometer-scale images of hepatic and cerebral glycogen formation following the administration of 13C-labeled glucose into awake animals. This is made possible through the combined use of electron microscopy (EM) and ultra-high spatial resolution ion microprobe (NanoSIMS) imaging, which provides maps of isotopic ratios on thin-sections of biological tissue with a spatial resolution of around 100 nm. 13,14 This allows both the precise localization of glucosyl residues within subcellular compartments and quantitative isotopic (here 13C/ 12C) ratio determination, providing the capability to trace exactly where and when in the compartments of single cells new glycogen deposits are formed during the isotopic labeling experiment.
Methods Animal preparation and injection protocol All experiments were conducted in accordance with relevant guidelines and regulations and approved by the local ethics committee (office véterinaire du canton de Vaud). Adult (10-24 week-old) wild-type NMRI male mice were fasted overnight prior to each experiment (n = 8). A 1.1 M aqueous [U- 13C]glucose solution (99% 13C isotopic enrichment, SigmaAldrich, Buchs, Switzerland) was prepared and a series of doses corresponding to 1 mg/g were injected intraperitoneally (i.p.) in awake mice at time intervals of 0, 10, 20, 60, 90, 120 and 150 min. The animals were placed in their cage after each injection. Blood samples were taken before each injection to monitor the time evolution of the glucose plasma level. At different time points (1, 2, and 3 h, respectively), after the initial injection, mice were deeply anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally injected) and transcardially perfused with 2 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 M phosphate buffer at pH 7.4. 15 All procedures involving the animals were performed using humane methods.
Tissue sample preparation for EM and NanoSIMS The brain and liver of each mouse were removed 2 h after perfusion and 70-μm-thick vibratome (Leica VT1200, Leica Biosystems, Nussloch, Germany) sections were cut through the liver and in the coronal plane through the neocortex. The sections were then washed in cacodylate buffer (0.1 M, pH 7.4), postfixed for 40 min in 1.5% potassium ferrocyanide and 1% osmium tetroxide, followed by 40 min in 1% osmium tetroxide alone, and 40 min in 1% uranyl acetate in water. They were then dehydrated through a series of increasing concentrations of ethanol, and infiltrated with Durcupan resin (Fluka, Buchs, Switzerland). The sections were flat embedded between glass slides in fresh resin and left for 24 h at 60 °C for the resin to harden. Thin sections at 70 nm thickness were cut with the diamond knife and collected on formvar coated alphanumeric grids or single slot grids with a pioloform support film. EM imaging Images of brain and liver ultra-thin section were recorded in a transmission electron microscope (Tecnai Spirit, FEI Company, Eindhoven The Netherlands) at 80 keV with a CCD camera (4 k × 4 k Eagle cameria, FEI Company). Individual EM images were collaged together using the Photoshop software (Adobe Systems, San Jose, CA, USA). Images were placed into different layers of a single file and one by one their transparency was increased to view the image below. This layer was then moved manually until its correct alignment was seen. This was repeated for all layers until the final collaged image was complete. Quantitative NanoSIMS isotopic imaging To image and quantify in situ the subcellular distribution of 13Cenriched glycogen within liver and brain cells, the exact same areas imaged by EM were analyzed with a NanoSIMS 50 L ion microprobe (CAMECA SAS, Gennevilliers, France), enabling direct correlation of ultrastructural (EM) and isotopic (NanoSIMS) images. EM grids were mounted on 10-mm diameter aluminum stubs and coated with about 10 nm gold. They were bombarded with a 16 keV primary ion beam of Cs + (1-3 pA) focused to a spot size of about 100-150 nm on the sample surface. Secondary molecular ions 12 14 − C N and 13C 14N − were simultaneously collected in electron multipliers at a mass resolution sufficient to avoid potentially problematic isobaric interference of 12C 15N − on 13C 14N −. Charge compensation was not necessary. Isotopic images ranging in size between 15 × 15 μm and 50 × 50 μm containing 256 × 256 pixels were obtained by rastering the primary beam across the sample surface with a dwell-time of 5 ms. Images were processed using the L’IMAGE® software (Larry R. Nittler, Carnegie Institution of Washington, Washington, DC, USA). 13 12 C/ C ratio distribution maps were obtained by taking the ratio between the drift-corrected 13C 14N − and 12C 14N − images. 13Cenrichments were expressed in the conventional delta notation: δ C ð‰Þ ¼ 13
C mes −1 1000; C nat
where Cmes is the measured 13C/ 12C ratio and Cnat is the average natural 13C/ 12C ratio measured regularly in non-labeled, identically
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Figure 1. Nanometer scale imaging of liver glycogen synthesis from [U- 13C]glucose. (A) EM image of liver thin section prepared from a fasted mouse 60 min (top) and 120 min (bottom) after the first injection of [U- 13C]glucose, respectively; yellow arrowheads point to glycogen granules; (B) Quantitative NanoSIMS 13 C-enrichment map of precisely the same region on the same thin sections; (C) 13C-enrichment profile along the line drawn on the NanoSIMS image in (B).
prepared samples throughout the period of NanoSIMS analyses (0.01104 ± 0.00004 (1 S.D.), n = 12). NanoSIMS isotopic maps were overlaid onto the EM images, using Photoshop software. The individual 12C 14N − images, which define cellular structures, such as the nucleus and mitochondria were initially overlaid to achieve an accurate correspondence between the NanoSIMS and EM field of view. Then individual NanoSIMS 13C/ 12C ratio maps were placed in the same position as their corresponding 12C 14N − images, which were subsequently removed. The 13C/ 12C ratio maps were then made 50% transparent to simultaneously view the underlying EM images. Statistical analysis Statistical analyses were performed by using the OriginPro 9.0G software (OriginLab, Northampton, MA, USA). Kolmogorov– Smirnov first tested the data for normality. Student’s t test was used for comparing groups of data. Results were considered significant at 5%. Throughout the paper, values given are mean ± S.D., and error bars show S.D.
Results The fixation, staining and embedding protocol used in this experiment is a conventional method for preparing tissue for electron microscopy, and clearly shows the cell ultrastructure, including the glycogen granules within both the brain and liver.
These are densely stained particles with a diameter of 10-40 nm, often clustered into larger aggregates (Figure 1, A). The spatial distribution of the 13C/ 12C isotopic ratio was imaged by NanoSIMS over the same area viewed in the EM in brain and liver sections (see e.g. Figure 1, B). Overlays of the NanoSIMS and EM images show local isotopic enrichments associated with the glycogen granules. It also gives the possibility to quantitatively determine the spatial variation of the 13 C enrichment along specific directions within the sections (see Figure 1, C). Spatially-resolved time evolution of glycogen synthesis in hepatocytes Large areas (minimum of 90 × 90 μm 2) of liver tissue from each experiment were imaged with EM, as illustrated in Figure 2, A. The glycogen granules appeared to be homogenously distributed among the hepatocytes identified in the larger collaged EM views. In these images, the localization of individual cell nuclei (highlighted by white lines in Figure 2, A) and plasma membranes (blue lines in Figure 2, A) is easily distinguished, as are glycogen aggregates. Strong heterogeneities in the spatial distribution of the isotopic ratio were observed and clear shifts from the natural 13C/ 12C ratio were detected in all liver sections obtained from mice exposed to [U- 13C] glucose for 60 min or longer (Figures 1, B and 2, A). Divisions between the perinuclear region surrounding the nucleus, and the peripheral region adjacent to the cell membrane (Figure 2, A) showed that the labeling is initially stronger in the
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Figure 2. Mapping the subcellular distribution of glycogen formation. (A) EM (left column) and 13C/ 12C NanoSIMS (right column) images of hepatocytes after 60, 120, and 180 min of exposure to [U- 13C]glucose, revealing the spatiotemporal progression of glycogen formation; white contours outline the hepatocyte nuclei, blue contours outline the hepatocyte membranes, and red contours divide each hepatocyte into a perinulear (i.e. close to nucleus) and a peripheral region; Small red and blue circles outline glycogen occurrences in the perinuclear and peripheral regions of each hepatocyte, respectively; (B) Histogram comparing 13 C-enrichment of liver glycogen located in two different regions of the hepatocytes; The initial (first 120 min) preferential region of enrichment, namely the perinuclear region, is later (180 min) labeled at about the same level as the peripheral region,.
perinuclear region (Figures 1, C and 2, B) in the time series analysis. The isotopic enrichment of glycogen aggregates located within both the perinuclear and peripheral regions of the
hepatocytes was determined from the NanoSIMS analyses and plotted as a function of the duration of the labeling period (Figure 2, B).
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Figure 3. Brain glycogen synthesis from [U- 13C]glucose. (A) EM image of brain thin section prepared from a fasted mouse 180 min after the first injection of [U- 13C]glucose; black arrows point to glycogen granules; (B) Quantitative NanoSIMS 13C-enrichment map correlated with the EM image; (C) 13C-enrichment profile along the line drawn on the NanoSIMS image in (B).
Spatial distribution of
13
C-labeled cerebral glycogen
In EM images of thin sections through the entire cortical thickness, glycogen granules were unambiguously identified (black arrows in Figure 3) and were exclusively found in the cytoplasm of astrocytes. 16 As expected from earlier studies, the density of glycogen aggregates was much lower in brain tissue than in liver tissue. All glycogen aggregates detected in astrocytes exhibit a 13C/ 12C ratio above the natural isotopic ratio. As observed in hepatocytes, the glycogen granules are not homogeneously labeled. However, there was no specific labeling pattern. On average, in animals euthanized after 3 h of labeling, the isotopic enrichment of glycogen was much lower in brain slices than in liver slices, with mean values of δ 13C = 170 ± 140 ‰ compared to δ 13C = 4220 ± 700 ‰, respectively (Figure 4). From these data, it is apparent that the 13C-enrichment observed in brain glycogen after 3 h is about 25 times lower than in the liver. Discussion The experiments presented here demonstrate how it is possible to study the incorporation of glucose into hepatic and cerebral glycogen at high resolution. Although it is known
that tissue glycogen content might decrease due to degradation during the standard preparation steps used in the present work, 17 the carbon isotopic ratio maps will not be affected if such loss affects glycogen granules in a homogeneous fashion. In vivo NMR studies have used similar doses of 13C-labeled glucose to study glycogen metabolism in rodents and it is therefore possible to compare these studies with the results obtained here. A liver glycogen 13C-enrichment of about 11 000 ‰ (i.e. a factor of 12) compared to the natural 13C/ 12C ratio of 0.011 was measured after 3 h in rats that were injected 6 mg/g [1- 13 C]glucose. 7 This is about twice the mean enrichment deduced from our data: 4220 ± 700 ‰ observed in liver glycogen aggregates after 3 h following the injection of 7 mg/g [U- 13C]glucose. However, our study used [U- 13C]glucose instead of [1- 13C]glucose and most labeled glucose-6-phosphate (glucose-6-P) that is incorporated into glycogen is formed from [U- 13C]pyruvate molecules deriving from glycolysis since it has been demonstrated that gluconeogenic compounds are the major carbon source for hepatic glycogen. 18 A substantial fraction of the 13C is thus lost in the form of 13CO2. 19,20 With regard to our NanoSIMS analyses, it must also be considered that 12C ions originating from the embedding material (in particular epoxy resin) introduced during sample preparation will to some extend contribute to a lowering of the apparent isotopic enrichments.
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Figure 4. Incorporation of 13C from [U-13C]glucose into astrocytic and hepatocytic glycogen. (A) Schematic representation of the incorporation of 13C into liver and brain glycogen from blood [U-13C]glucose; (B) Average isotopic enrichment in liver (orange) and brain (blue) glycogen aggregates after 3 h labeling through i.p. injection of [U-13C]glucose. The observed mean value was δ13C = 170 ± 140‰ and δ13C = 4220 ± 700‰ in the brain and the liver, respectively.
Overall, although there is a relatively large standard deviation on the mean enrichment from NanoSIMS analyses of small parts of the liver, the 13C-enrichment we observe is comparable to the enrichment obtained from the in vivo 13C NMR analyses. The fact that the average 13C/ 12C ratio determined with NanoSIMS in brain glycogen after 3 h is about 25 times lower than the ratio measured in liver glycogen is also in good agreement with earlier in vivo 13C NMR studies. Indeed, the glycogen turnover rate of ~0.5 μmol⋅g −1⋅h −1 previously observed in rat brain by 13C NMR21 corresponds to a rate that is about 30 times lower than the 15 μmol⋅g − 1 ⋅h −1 deduced from the rate of net glycogen synthesis and
degradation measured in rat liver. 5 The general consistency with the in vivo 13C NMR observations supports the fact that the NanoSIMS images capture and correctly quantify glycogen formation, even if some of the glycogen might be lost in the sample preparation. Therefore, the proportion of glycogen that might be lost through sample preparation and/or rapid metabolic degradation associated with cell death was similar in brain and liver tissue. In addition, if the 13 C incorporation into glycogen follows a last-in first-out scheme, as expected from earlier studies, 22,23 only a relatively small fraction of glycogen was lost because the relative 13C-enrichment was comparable to that measured in vivo by 13C NMR. If we nevertheless assume that a considerable part of brain glycogen was lost during cell death, we must conclude that the glucosyl residues that were labeled within the 3 h of the experiment are not preferentially the ones used, via glycogenolysis, as emergency energy reserve. Our data demonstrate that initial glycogenesis from 13C-labeled glucose molecules injected into fasted animals is taking place with a strong preference for the perinuclear region of hepatocytes. This observation is in contrast to previous in vitro experiments performed using [ 14C]glucose combined with EM, 24,25 where it was observed that glycogen synthesis starts at the cell periphery, i.e., near the plasma membrane. This discrepancy may simply be a reflection of differences in the way in vitro and in vivo cells handle glucose. In the in vitro studies, it was shown, as expected, that the 14 C-label is highly correlated with glycogen synthase concentration, which was not measured in the present study. The early and strong presence of 13C-labeled glycogen in the perinuclear region observed in our study is itself consistent with the fact that glucokinase is localized in the nucleus of hepatocytes when glucose level is low, before being released into the cytosol as soon as the blood glucose level increases. 26,27 No significant deviation from the natural fractional 13C/ 12C ratio was observed inside the hepatocyte nuclei, and if glycogen was present as suggested in an earlier study, 28 it was around or below the detection limit of the NanoSIMS, which is about 50 ‰, possibly because the turnover rate was much slower than inside the cytosol. Abnormal glycogen metabolism has been associated with disorders affecting the central nervous system. Inability to inhibit glycogen synthesis in neurons is the basis of diseases such as Lafora disease, 29 and impaired activity of glycogen branching enzyme occurs in adult polyglucosan body disease. 30 Furthermore, it is now well established that glycogen turnover is altered in many types of tumor cells. 31 Nanometer scale imaging of glycogen metabolism represents a powerful new tool for investigating glycogenassociated disorders at an entirely new level of resolution. Our results open the way for new types of metabolic imaging that will provide fundamental insights into cellular use of a variety of metabolic substrates in both healthy and diseased mammalian tissue. Acknowledgments We thank Dr. Céline Loussert (Electron Microscope Facility at University of Lausanne) for expert advice and technical assistance.
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