Volume 147, Issue 2 pp. 190-203

Original Article

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RS1 (Rsc1A1) deficiency limits cerebral SGLT1 expression and delays brain damage after experimental traumatic brain injury

Anne Sebastiani,

Anne Sebastiani

Department of Anesthesiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

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Frederik Greve,

Frederik Greve

Department of Anesthesiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

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Christina Gölz,

Christina Gölz

Department of Anesthesiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

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Carola Y. Förster,

Carola Y. Förster

Department of Anesthesiology, University of Würzburg, Würzburg, Germany

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Hermann Koepsell,

Hermann Koepsell

orcid.org/0000-0001-5118-7088

Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany

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Serge C. Thal,

Corresponding Author

Serge C. Thal

[email protected]

orcid.org/0000-0002-1222-8729

Department of Anesthesiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

Address correspondence and reprint requests to Dr Serge C. Thal, Department of Anesthesiology, University Medical Center of the Johannes Gutenberg-University, Langenbeckstrasse 1, 55131 Mainz, Germany. E-mail: [email protected]Search for more papers by this author

Anne Sebastiani,

Anne Sebastiani

Department of Anesthesiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

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Frederik Greve,

Frederik Greve

Department of Anesthesiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

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Christina Gölz,

Christina Gölz

Department of Anesthesiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

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Carola Y. Förster,

Carola Y. Förster

Department of Anesthesiology, University of Würzburg, Würzburg, Germany

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Hermann Koepsell,

Hermann Koepsell

orcid.org/0000-0001-5118-7088

Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany

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Serge C. Thal,

Corresponding Author

Serge C. Thal

[email protected]

orcid.org/0000-0002-1222-8729

Department of Anesthesiology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

First published: 19 July 2018

https://doi.org/10.1111/jnc.14551

Citations: 15

These authors contributed equally to this work.

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Abstract

Acute cerebral lesions are associated with dysregulation of brain glucose homeostasis. Previous studies showed that knockdown of Na⁺-D-glucose cotransporter SGLT1 impaired outcome after middle cerebral artery occlusion and that widely expressed intracellular RS1 (RSC1A1) is involved in transcriptional and post-translational down-regulation of SGLT1. In the present study, we investigated whether SGLT1 is up-regulated during traumatic brain injury (TBI) and whether removal of RS1 in mice (RS1-KO) influences SGLT1 expression and outcome. Unexpectedly, brain SGLT1 mRNA in RS1-KO was similar to wild-type whereas it was increased in small intestine and decreased in kidney. One day after TBI, SGLT1 mRNA in the ipsilateral cortex was increased 160% in wild-type and 40% in RS1-KO. After RS1 removal lesion volume 1 day after TBI was reduced by 12%, brain edema was reduced by 28%, and motoric disability determined by a beam walking test was improved. In contrast, RS1 removal did neither influence glucose and glycogen accumulation 1 day after TBI nor up-regulation of inflammatory cytokines TNF-α, IL-1β and IL-6 or microglia activation 1 or 5 days after TBI. The data provide proof of principle that inhibition or down-regulation of SGLT1 by targeting RS1 in brain could be beneficial for early treatment of TBI.

Abbreviations used

BBB: blood-brain barrier
CCI: controlled cortical impact
dpi: days post injury
GLUT: glucose transporter
Iba-1: ionized calcium binding adaptor molecule-1
MCAO: middle cerebral artery occlusion
PPIA: cyclophilin A
qPCR: quantitative real-time polymerase chain reaction
RT-PCR: real-time reversely transcribed polymerase chain reaction
SEM: standard error of the mean
SGLT: sodium glucose cotransporter
TBI: traumatic brain injury

The brain is highly dependent on the availability of glucose and oxygen to maintain basic cellular function and neuronal activity. Glucose is the major substrate for energy producing pathways in glial and neuronal cells (Magistretti and Pellerin 1999). Securing of energy supply in pathological scenarios is of utmost importance for the maintenance of neuronal activity and prevention of neuronal lesions. Acute pathologic events such as traumatic brain injury (TBI) and stroke induce severe metabolic crises in the brain (Bergsneider et al. 1997; Bartnik-Olson et al. 2013). In such situations injury-induced ionic and neurochemical cascades increase energetic requirements. This is counteracted by hyperglycolysis leading to an increase in glucose demand (Goodman et al. 1999). During brain trauma and stroke blood glucose and glucose content in the affected brain areas increase. This hyperglycemia is correlated with negative clinical outcome (De Salles et al. 1987; Lam et al. 1991; Rovlias and Kotsou 2000; Pasternak et al. 2008; Harada et al. 2009; Liu-DeRyke et al. 2009). An up-regulation of facilitative-diffusion glucose transporters of the glucose transporter (GLUT) family during ischemia and trauma has been described (Lee and Bondy 1993; Cornford et al. 1996; Urabe et al. 1996; Hamlin et al. 2001).

In mammals solute transporters from two transporter families mediate the transfer of glucose across plasma membranes of the blood-brain barrier, glial cells, and neurons, the facilitative glucose transporters of the GLUT (SLC2) (Maher et al. 1994) family and the sodium glucose cotransporters of the SGLT (SLC5) (Poppe et al. 1997; Scheepers et al. 2004; Wright et al. 2011; Harada et al. 2013) family. Glucose transport in brain tissue was originally attributed to sodium-independent GLUT transporters, GLUT1 which is mainly expressed in endothelial cells of the blood-brain barrier (BBB) and glial cells, and GLUT3 which is expressed in neurons (Maher et al. 1994; Hamlin et al. 2001; Ferreira et al. 2011). More recently it has been shown that also members of the SGLT family are expressed in the brain.

Most importantly the expression of the sodium-D-glucose cotransporter SGLT1 has been demonstrated and its functional relevance under physiological and pathophysiological conditions has been discussed (Poppe et al. 1997; Yu et al. 2013; Yamazaki et al. 2015, 2017; Koepsell 2017). Immunocytochemical localization and in situ-hybridization experiments strongly suggest that SGLT1 is expressed in neurons and provide hints on additional locations of SGLT1 in glial cells and the BBB (Poppe et al. 1997; Elfeber et al. 2004; Vemula et al. 2009; Yu et al. 2010; Yamazaki et al. 2015). Unfortunately, the immunocytochemical localizations of SGLT1 in brain must be considered with caution because using Sglt1^−/− mice as specificity control for the immunolocalization of SGLT1 in mouse brain indicate that the so far employed antibodies do not allow a specific identification of SGLT1 (Madunic et al. 2017). A pathophysiological role of (a) SGLT transporter(s) during energy deficiency was demonstrated by showing in a rat model that an uptake of the SGLT-specific glucose analog methyl-α-D-glucopyranoside was increased in an epileptic focus (Poppe et al. 1997) and that the lesion volume after middle cerebral artery occlusion (MCAO) mimicking stroke was decreased in the presence of SGLT-specific inhibitor phlorizin (Vemula et al. 2009; Yamazaki et al. 2012). Employing SGLT1 knockdown in mouse brain, a critical role of SGLT1 for development of infarct size and clinical symptoms after MCAO was demonstrated (Yamazaki et al. 2015). The authors also reported that the immunoreactivity in Western blots of commercial antibody raised against SGLT1 was increased after MCAO.

In the present study, we investigated the effects of TBI on SGLT1 mRNA abundance in absence and presence of regulatory protein RS1. RS1 is an intracellular 67–68 kDa protein that is coded by the intronless gene RSC1A1 on chromosome 1 and is expressed in various organs including small intestine, kidney, and brain (Veyhl et al. 1993; Koehler et al. 1996; Lambotte et al. 1996; Poppe et al. 1997). RS1 regulates SGLT1 on transcriptional and post-translational levels. Post-translational regulation of SGLT1 was extensively investigated in vitro and was demonstrated in tissue of the small intestine (Veyhl et al. 2006; Chintalapati et al. 2016; Veyhl-Wichmann et al. 2016), whereas transcriptional regulation was demonstrated in the renal cell line LLC-PK₁ (Korn et al. 2001). In mice in which RS1 was removed (RS1^−/− mice), we observed that plasma membrane abundance of SGLT1 in small intestine was increased to a similar level as after glucose-induced post-translational up-regulation (Osswald et al. 2005; Veyhl-Wichmann et al. 2016). We investigated whether up-regulation of SGLT1 is observed after TBI focusing on the analysis of SGLT1 mRNA expression. Employing RS1^−/− mice we investigated whether RS1 removal changes expression of SGLT1 mRNA in small intestine, kidney, and brain and influences cerebral SGLT1 mRNA expression after TBI.

Surprisingly we detected that RS1 removal did not alter SGLT1 mRNA in brain whereas SGLT1 mRNA abundance in small intestine and kidney was increased and decreased respectively. After CCI in wild-type mice, SGLT1 mRNA abundance in the lesioned brain area transiently increased 2.6-fold whereas in RS1-KO mice it was only increased by 40%. In the RS1-KO mice the volume of lesioned tissue, ipsilateral brain edema, and motoric disability were reduced whereas no significant effects on expression of inflammation markers and activation of glial cells were observed. The data indicate that RS1 is involved in the regulation of SGLT1 in brain and that this regulation has impact on the outcome after TBI.

Methods

Experimental animals

In the present study, 139 male C57BL/6 mice (RRID:IMSR_CRL:27; Charles River, Sulzfeld, Germany) and RS1 knockout mice on a C57BL/6 background (RRID:MGI:3526863; RS1^−/− mice), lacking the regulator gene Rsc1A1 (Osswald et al. 2005) and wild-type litter mates were investigated (weight: 24.9–47.9 g). RS1 deficiency was confirmed by quantitative real-time polymerase chain reaction (qPCR) on the mRNA and by genotyping at the DNA level. Mice were kept under 12 : 12 light and dark cycle with free access to food and water. All performed experiments were approved by the Animal Ethics Committee of the Landesuntersuchungsamt Rheinland-Pfalz, Germany (protocol number 23177-07/G11-1-007). Before and during experiments animal care was in accordance with the guidelines of the Johannes Gutenberg-University, Mainz, Germany. The presentation of the experiments is in accordance to the ARRIVE guidelines. The study was not pre-registered. Randomization was performed by a third person not associated with the study using a web-based random group generator (http://www.pubmed.de/tools/zufallsgenerator). The following exclusion criteria were applied: severe weight loss (> 15%) in combination with pain symptoms (tooth crunches) or severe behavioral deficits (decreased locomotion, convulsions, stupor). In the present study no animals were excluded because of these criteria. Pain and stress level were determined in a separate set of experiments showing limited stress compared to other cerebral injures, e.g. subarachnoid hemorrhage (data not shown). Attributed to these data no pain medication was given to these animals. The study includes different experiments including time course investigations, which are summarized in a schematic representation of the experimental timeline and groups (Fig. 1).

Details are in the caption following the image — **Figure 1**
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Schematic representation of the experimental timeline. Graphical presentation of experiments and outcome parameter with timeline (dpi = days post injury) from controlled cortical impact (CCI) to tissue or blood harvesting (sampling).

Experimental traumatic brain injury

All experiments were performed at day-time (7 am until 7 pm). Animals were anesthetized with isoflurane (induction: 4 vol%; maintenance: 2 vol%) in an oxygen/air mixture (40%O₂/60% N₂) via face mask and placed into a stereotactic frame. Anesthesia protocol was selected based on data from previous studies comparing different anesthesia protocols during experimental TBI (Luh et al. 2011; Thal et al. 2012). Core temperature was kept constant at 37°C using a feedback-controlled heating system (Hugo Sachs, March-Hugstetten, Germany). Trauma was induced by controlled cortical impact (CCI) as described before (Thal et al. 2013): After craniotomy on the right rostro-caudal plane a mechanical lesion was induced on the right parieto-temporal cortex by a custom fabricated impactor (L. Kopacz, Germany) with following parameters: tip diameter of 3, 1.5 mm brain penetration, impact duration of 150 ms, and impact velocity of 8 M/s. Following CCI the craniotomy was sealed with tissue glue (PNZ# 02330167; Histoacryl; B Braun Melsungen AG, Melsungen, Germany). The skin was sutured closed. Animals were placed to their individual home cages in an incubator (33°C, 35% humidity; IC8000, Dräger, Lübeck, Germany) for 2 h.

Histological evaluation of secondary brain damage

Animals were euthanized by cervical dislocation under isoflurane anesthesia (4 Vol % for 1 min.). Brains were quickly removed, snap frozen on powdered dry ice, and stored at −20°C. Brains were cut in the coronal plane using a cryostat (HM 560 Cryo Star, Thermo Fisher Scientific, Walldorf, Germany). Sections (10 μm) were collected at 500 μm interval and stained with cresyl violet. For the quantification of lesion volume, areas of both hemispheres and the injured brain tissue were measured using a computerized image system (Delta Pix Insight, Delta Pix, Maalov, Denmark) by an investigator blinded to the randomization. Lesion volumes were calculated by multiplying lesion areas obtained from 16 consecutive sections with the distance-interval of 500 μm [0.5 * (A₁ + A₂ + A₃ + … + A_n)] (Thal et al. 2013).

Gene expression (mRNA) analysis

For time series analysis tissue preparation was performed as follows: Brains were quickly removed and placed in a cooled brain matrix (Cat# BSMAA001-1, Zivic Instruments, Pittsburgh, WA, USA). For all other studies samples of perilesional injured brain tissue were collected during the cryosectioning procedure (−20°C). Brain tissue was stored at −80°C until processing. Quantification of mRNA was performed by real-time reversely transcribed polymerase chain reaction as previously described (Thal et al. 2008). Absolute copy numbers of target genes were normalized against cyclophilin A (PPIA) as housekeeping gene. Applied primers and probes are shown in Table 1. Same amounts of cDNA were amplified in duplicates using Kapa Probe Fast qPCR (Cat# KK4703; peqlab biotechnology GmbH, Erlangen, Germany) for PPIA, LightCycler^® 480 Probes Master (Cat# 04887301001; Roche Deutschland Holding GmbH, Grenzach-Wyhlen, Germany) for IL-1β, IL-6, ABsolute™ Blue qPCR SYBR Green Mix (Thermo Fisher Scientific GmbH, Dreieich, Germany) for TNF-α and Absolute™ Fast qPCR Mix (Thermo Fisher Scientific GmbH) for SGLT1 and GLUT1, and Maxima Probe qPCR Mastermix (Thermo Fisher Scientific GmbH) according to the manufacturer's instructions.

Table 1. Primers and probes for real-time PCR

Gene (amplicon size, annealing temp)

Oligonucleotide sequence (5′-3′)

Accession No.

Cyclophilin A (PPIA)

(146 bp, 55°C)

Forw: 5′-GCGTCTSCTTCGAGCTGTT-3′

Rev: 5′-RAAGTCACCACCCTGGCA-3′

FL: 5′-GCTCTGAGCACTGGRGAGAAAGGA-FL

Cy5: Red-TTGGCTATAAGGGTTCCTCCTTTCACAG-Phos

NM_008907

Rsc1a1 (RS1)

(391 bp, 55°C)

Forw: 5′-CAGGAAAGACAGAAATCGTAGG-3′

Rev: 5′-GGAAAGATGAACGGTGGAAG-3′

FL: 5′-TGAAGGTCTGGGTGATGGCTTGTC-FL

Cy5: Red-TGACCGAGAAGATGTCCGCAGA-Phos

NM_019810.4

Slc5a1 (SGLT1)

(154 bp, 55°C)

Forw: 5′-CAGAAATACTGCGGCACAC-3′

Rev: 5′-CCATCATGACAGACAGCATCA-3′

FL: 5′-TGAGTTCCACCACGAGCGT-FL

LC670: Red-GGGTAGGCGATGTTGGTACAGCC-Phos

NM_023544.5

Slc2a1 (GLUT1)

(98 bp, 55°C)

Forw: 5′-CCCAGAAGGTTATTGAGGAGTT-3′

Rev: 5′-GGAGAGAGACCAAAGCGTG-3′

FL: 5′-CATGGAACCACCGCTACGGA-FL

LC670: Red-AGCCCATCCCATCCACCACAC-Phos

NM_011400

IL-1β

(348 bp, 55°C)

Forw: 5′-GTGCTGTCGGACCCATATGAG-3′

Rev: 5′-CAGGAAGACAGGCTTGTGCTC-3′

FL: 5′-TAATGAAAGACGGCACACCCACCC-FL

Cy5: Red-CAGCTGGAGAGTGTGGATCCCAAGC-Phos

NM_008361

TNF-α

(212 bp, 62°C)

Forw: 5′-TCTCATCAGTTCTATGGCCC-3′

Rev: 5′-GGGAGTAGACAAGGTACAAC-3′

NM_008361

IL-6

(141 bp, 55°C)

Forw: 5′-GAGGATACCACTCCCAACAGACC-3′

Rev: 5′-AAGTGCATCATCGTTGTTCATACA-3′

NM_031168

Rsc1a1: Regulatory solute carrier protein, family 1, member 1 (Mus musculus), SLC5a1: Solute carrier family 5 (sodium/glucose cotransporter), member 1

Quantification of brain water content

Animals were sacrificed at 24 h after CCI under deep anesthesia with isoflurane. Percentage brain water content was analyzed using speed vacuum-drying method by wet weight-to-dry weight ratio. Brains were quickly removed and placed in a brain matrix. Damaged and pericontusional brain tissue was cut into sections (3 mm thickness; separated by the middle of the damage). The left and right side were separated along the anatomic midline. Only samples from the upper quadrant of these sections (approximately 3 mm³) were used for analysis and the weight was determined before and after drying by a centrifugal vacuum concentrator as recently described in detail (Sebastiani et al. 2017). In brief, we put samples into preweighed plastic tubes (Eppendorf AG, Hamburg, Germany) and sealed these with a cap. After determining the wet weight (Scaltec SBA 32; Denver Instruments, Bohemia, NY, USA) we placed the opened tubes in a vacuum centrifuge for 72 hours to determine the dry tissue weight. Percentage of brain water content was calculated by the following equation: Brain water content [%] = 100 × wet weight – dry weight/wet weight.

Determination of motoric disability by a beam walking test

Functional outcome before and after CCI was determined by an investigator which was blinded to the allocation of experimental groups. In the beam walking test, the ability of the mice to traverse three 50 cm long, square cross-section wood beams with widths of 1, 2, and 3 cm. Beam walking tests were performed 1 day before CCI and one, three, and five days after CCI. Healthy mice were successful in all tasks and received a score of 0. Walking pattern with more than four feet misplacements was rated with one point, inability to walk with two points. The maximum number of points (failure to walk on all three beams) was six.

Quantification of brain glucose and glycogen content

Ipsilateral and contralateral brain tissues samples (upper quadrant) were homogenized (Retsch MM300, Germany; 20 Hz, 60 s) in 200 μL ice-cold 6% perchloric acid containing 1 mM EDTA for 2 × 60 s. The homogenates were placed for 5 min in a boiling water bath. For tissue glycogen content, glycogen was hydrolyzed by incubation for 2 h at 20–25 °C with 500 μL of 50 μg/mL amyloglucosidase in 50 mM sodium acetate buffer containing 0.02% bovine serum albumin and neutralized with a KOH compound solution (3 M KOH, 0.3 M imidazole, and 0.4 M KCl) to pH 5.5–7.5. After centrifugation at 20.817 g for 10 min at 4°C, supernatants were taken to determine glucose content. Glucose content was determined using a modified coupled enzyme assay method (Passonneau and Lauderdale 1974) and D-glucose UV-method kit (Cat# 10716251035; Boehringer Mannheim, R-Biopharm AG, Germany). Measurements of NADPH were performed in a fluorescence plate reader (Progema, GloMax multi detection system, Fitchburg, MA, USA) at Ex/Em 365 nm/420–460 nm. Tissue glycogen levels indicated as glucose units were calculated by subtracting the final concentration of glucose per g wet weight of the non-hydrolyzed tissue sample from the concentration of glucose per g wet weight of the hydrolyzed tissue sample.

Microglia staining

Microglial cells expressing ionized calcium binding adaptor molecule-1 (Iba-1) were quantified by immunocytochemical staining sections. Therefore, cryosections were fixed in 4% paraformaldehyde, rinsed with PBST, phosphate-buffered saline (PBS) with 0.3% TritonX (Cat# 93426; Sigma, St. Louis, MO, USA), and incubated in blocking solution consisting of PBS with 5% normal goat serum (DAKO, Glostrup, Denmark). Antibodies were solved in PBST. At 20–25 °C sections were incubated over night with rabbit anti-Iba-1 antibody (1 : 1500; Cat# 019-19741, RRID: AB_839504; WAKO Pure Chemical Industries, Osaka, Japan) (Ito et al., 1998). After rinsing biotinylated anti-rabbit IgG (H+L) (Cat# BA-1000; Vector Laboratories Inc., Burlingame, CA, USA) was used as secondary antibody. A peroxidase, ABC-Complex (Cat# PK-4000; Vector Laboratories Inc., Burlingame, CA, USA), was applied after rinsing. The non-bound complex was washed out and then the chromogen DAB (DAKO, Glostrup, Denmark) was added. Then, a background staining with Mayer's hemalum solution (Cat# 254766; AppliChem GmbH, Darmstadt, Germany) was performed. Total number of positive cells was counted in ipsi- and contralateral perilesional brain cortex (region of interest: 0.52 ×0.65 mm) at bregma −2.36 mm according to Mouse Brain Library atlas (RRID:SCR_001112; http://mbl.org) by an investigator blinded to group allocation.

Statistical analysis

All experiments were randomized performed by blinded investigators (computer-based randomization). To determine the required sample size, an a priori power analysis using G∗Power (RRID:SCR_013726) was performed using lesion volume data from previously published studies (Faul et al. 2009). The a priori power analysis was performed to determine an effect size of 0.7, standard statistical power (1-β) of Pβ=0.95 and a significance level (α) of 0.05. Statistical analysis was performed using GraphPad Prism 7 statistical software (RRID:SCR_002798; GraphPad Software Inc., La Jolla, CA, USA). For comparison of multiple groups one-way anova with post-hoc Holm-Sigak comparison was employed. For comparison between two groups significance was determined by Welch-t test. Values of p < 0.05 were considered significant. Data are presented as mean and standard error of the mean (SEM).

Results

RS1 Removal has different effects on SGLT1 mRNA abundance in small intestine, kidney and brain cortex

We confirmed the genotype of the employed RS1^−/− mice by showing that RS1 mRNA is absent in duodenum, kidney, brain cortex, and hippocampus (Fig. 2a–d). In brain about 20- and 10-fold lower concentrations of RS1 mRNA were observed compared to duodenum and kidney respectively. For the expression SGLT1 mRNA dramatic differences were observed between organs. The SGLT1 mRNA concentrations in brain cortex and hippocampus were 24 000- and 48 000-fold lower compared to small intestine respectively (Fig. 2e–h). In RS1^−/− mice the abundance of SGLT1 mRNA in duodenum was 59% higher compared to wild-type whereas SGLT1 mRNA abundance in kidney was 33% lower (Fig. 2e and f). At variance, abundance of SGLT1 mRNA in brain cortex and hippocampus in RS1^+/+ and RS1^−/− mice was similar. The data suggest tissue-specific regulation of SGLT1 transcription.

CCI increases SGLT1 mRNA after one day and RS1 mRNA after three days

To investigate whether TBI alters mRNA abundance of SGLT1 and/or RS1 in wild-type mice, mRNAs of SGLT1, RS1, and PPIA were quantified in perilesional cortical brain tissue one, three, and five days after CCI and in a corresponding cortical region of untreated mice. Although the mRNA abundance of the housekeeping gene PPIA was similar in all groups (see legend of Fig. 2) the abundance of SGLT1 mRNA showed a transient about 2.5-fold increase 1 day after CCI (see Figs 3a and 4a). At variance, a 50% transient increase of RS1 mRNA was observed 3 days after CCI (Fig. 3b). The data suggest that transient post-traumatic up-regulation of SGLT1 transcription is followed by a less pronounced transient transcriptional up-regulation of RS1.

Removal of RS1 mitigates up-regulated SGLT1 mRNA after CCI

We compared mRNA abundance of SGLT1 (Fig. 4a) and GLUT1 (Fig. 4b) in RS1^+/+ and RS1^−/− mice which were untreated or underwent CCI. One day after CCI the abundance of SGLT1 mRNA was increased by 160% in wild-type whereas it was only increased by 52% in RS1^−/− mice (p < 0.01/0.05). Five days after CCI the mRNA increase was reversed to similar levels in both RS1^+/+ and RS1^−/− mice which were in the range of the values observed in untreated animals. Expression of GLUT1 mRNA was similar in untreated RS1^+/+ and RS1^−/− mice (Fig. 4b). The concentration of GLUT1 mRNA in brain cortex and hippocampus was 1000-fold higher compared to SGLT1. A trend for an about 20% increase 1 day after CCI was not significant, however, the mRNA concentrations determined 5 days after CCI were significantly lower compared to 1 day after CCI. The data suggest that RS1 is involved in the transient up-regulation of SGLT1 mRNA after TBI.

Mitigated up-regulation of cerebral SGLT1 mRNA after CCI after RS1-removal has no effect on cerebral glucose and/or glycogen concentrations

To determine whether the blunted up-regulation of SGLT1 mRNA expression after RS1 removal attenuates the increase of cerebral glucose and/or glycogen concentrations after TBI (Otori et al. 2004) we measured these compounds in wild-type and RS1^−/− 1 day after CCI in the ipsilateral and contralateral brain cortex (Fig. 5). The measurements after CCI were performed in ipsilateral and contralateral hemispheres. In wild-type and RS1^−/− mice similar glucose and glycogen concentration were determined. The cerebral glucose and glycogen concentrations in untreated mice were about 50% lower than the concentrations in the contralateral hemisphere of mice 1 day after CCI. After CCI the concentration of glucose in the ipsilateral hemisphere was 3-fold higher compared to the contralateral hemisphere whereas the concentration of glycogen in the ipsilateral hemisphere was fourfold higher. The data suggest that the reduced SGLT1 expression after CCI in RS1^−/− mice does not influence the cerebral increase of glucose and glycogen.

After removal of RS1 lesion volume and brain edema after CCI are mitigated

Next, we investigated the impact of RS1 expression on brain damage. RS1^−/− mice and corresponding wild-type littermates were subjected to CCI and lesion volumes and brain water content were determined after CCI. Lesion volume was assessed in Nissl-stained cryosections. One day after CCI the lesion volume in RS1^−/− animals was 12% smaller compared to wild-type mice (24.4 ± 1.3 mm³ vs. 27.8 ± 1.1 mm³, p < 0.05 (Fig. 6a). Five days after CCI the lesion volumes were decreased by more than 50% and no significant difference between RS1^−/− and wild-type mice was observed (12.1 ± 1.0 mm³ vs. 12.3 ± 0.6 mm³) (Fig. 6b). We also investigated whether the reduced lesion volume observed in RS1^−/− mice one day after CCI is correlated with reduced brain edema formation (Fig. 6c). The water content in brain of untreated RS1^+/+ and RS1^−/− mice was not significantly different (77.6 ± 0.2% vs. 77.9 ± 0.3%). One day after CCI the water content increased to 84.5 ± 0.4% in RS1^+/+ animals and to 82.9 ± 0.2% in RS1^−/− animals. The brain damage-induced 4.9% increase in water content in RS1^−/− mice was significantly smaller compared to brain damage-induced 6.8% increase observed in wild-type mice (p < 0.01). The data suggest that the transcriptional up-regulation of SGLT1 after TBI in brain has an impact on brain damage.

RS1 deficiency improves motoric impairment after CCI

To determine whether the reduced volume and edema after CCI observed in RS1^−/− mice is correlated with a mitigated motoric disability we performed beam walking tests 1 day before, and one, three and 5 days after CCI (Fig. 6d). Whereas RS1^+/+ and RS1^−/− animals managed the tasks before CCI (scores around 0) they showed slight motoric disabilities after CCI. Motoric impairment was most pronounced 1 day after CCI. Five days after CCI the impairment was diminished significantly (p < 0.01). Importantly, 1 day after CCI a significantly higher motoric disability was observed in RS1^+/+ mice compared to RS1^−/− mice (2.3 ± 0.4 points vs. 1.4 ± 0.3 points, p < 0.01). The data indicate that the effects of RS1 removal on SGLT1 up-regulation of SGLT1, lesion volume and edema after TBI are correlated with the clinical outcome.

RS1 deficiency does not alter expression of inflammatory cytokines, and microglia activation after CCI

To investigate the impact of RS1 deficiency on cerebral inflammation, mRNA expression of the inflammation related cytokines TNF-α, IL-1β, and IL-6 and the number of Iba-1 expressing microglial cells were quantified after CCI. Measurements were performed in wild-type and RS1^−/− mice without treatment and one or 5 days after CCI. Measurements after CCI were performed in perilesional and contralateral brain tissue. In wild-type and RS1^−/− mice similar mRNA concentrations of TNF-α, IL-1β, and IL-6 were observed (Fig. 7a–c). 50-fold up-regulation of TNF-α was observed 1 day after CCI which was slightly but significantly attenuated 5 days after CCI. One day and 5 days after CCI, IL-1β mRNA was up-regulated sixfold and threefold compared to untreated animals respectively. IL-6 mRNA was transiently up-regulated 40-fold compared to untreated animals showing not significant up-regulation 5 days after CCI. The data indicate that the decreased SGLT1 mRNA abundance 1 day after CCI observed after RS1 removal does not influence the up-regulation of mRNA expression of these cytokines.

To determine whether RS1 expression has an impact on the activation of microglia after traumatic brain injury we compared the number of microglial cells showing Iba-1 immunoreactivity between wild-type and RS1^−/− mice before and after CCI (Fig. 8a–d). One and five days after CCI, the number of cells with Iba-1 immunoreactivity was determined in a perilesional region and a corresponding contralateral region and in a corresponding cortical region of untreated animals. No differences between wild-type and RS1^−/− mice were observed. In the perilesional region, the number of cells with Iba-1 immunoreactivity increased slowly showing a 60% increase at 5 days after CCI. The data indicate that RS1 does not influence microglial cell activation.

Discussion

In the present study, we demonstrated transient up-regulation of sodium-D-glucose transporter SGLT1 mRNA in mice 1 day after CCI which was almost abolished when the intracellular SGLT1 regulator RS1 was removed. One day after CCI the lesion volume was smaller, the edema in the lesion region was reduced, and the motoric impairment was mitigated in RS1^−/− compared to RS1^+/+ mice. After CCI neither cerebral glucose and glycogen concentrations in the lesion area, nor cerebral expression of three inflammation-related cytokines, or microglia activation were changed after RS1 removal. The data support pathophysiological relevance of SGLT1 expression in brain during traumatic injury and identify SGLT1 and RS1 as potential drug targets for treatment of TBI.

Following TBI or brain ischemia disbalances of ionic and neurotransmitter distributions such as extracellular increase of potassium and glutamate, induce a largely raised energy demand. This is counteracted by a hyperglycolytic metabolic phase with high requirement for D-glucose that is followed by a period of hypoglycolytic metabolism (Bergsneider et al. 1997; Bartnik-Olson et al. 2013). After TBI circulating catecholamines increase and induce hyperdynamic cardiovascular response and an elevation of blood glucose which is correlated with increased glucose concentration in the injured brain area (De Salles et al. 1987; Lam et al. 1991; Thomas et al. 2000). The pathophysiological relevance of the blood glucose concentration became apparent from clinical studies which show that acute hyperglycemia is correlated with worsened clinical outcome (Widmer et al. 1992; Dietrich et al. 1993; Rovlias and Kotsou 2000; Jeremitsky et al. 2005; Liu-DeRyke et al. 2009). The elevated glucose concentration in injured brain tissue may influence water homeostasis by direct hygroscopic effects, which may cause early brain edema formation. Lesion of brain tissue is followed by expression of inflammation markers and activation of microglia (Chen et al. 2003; Zhang et al. 2006; Loane and Byrnes 2010; Jeong et al. 2013; Roth et al. 2014).

Trying to elucidate the pathophysiological mechanisms by which cerebral glucose after ischemia and TBI is increased, it was investigated whether the expression of GLUT1 in the BBB and glial cells and of GLUT3 in neurons was enhanced. Up-regulation of GLUT1 in the BBB and neurons after ischemia (Lee and Bondy 1993; Urabe et al. 1996) and of GLUT3 in neurons after ischemia and TBI (Urabe et al. 1996; Hamlin et al. 2001) has been reported. While this helped to explain the increase of cerebral glucose it does not account for the correlation of the elevated glucose concentration with poor clinical outcome which is mainly because of neuron degeneration (Chen et al. 2003). One involved mechanism may be that unphysiologically activated neurons are damaged by reactive oxygen species which are generated at high glucose concentrations (Li et al. 1999; Cook et al. 2008). Thus, modulation of oxidative stress after TBI has been shown to improve lesion size, edema formation, and clinical parameters (Lewen et al. 2000).

The pathophysiological role of SGLT1 during ischemia and TBI may be envisioned as follows. During slight brain ischemia and/or hypoglycemia, SGLT1 in brain vessels and/or neurons may be up-regulated and may help to secure energy supply (Poppe et al. 1997; Elfeber et al. 2004). Secondary active sodium-D-glucose cotransport by SGLT1 at low glucose concentrations is more efficient than glucose uptake by the passive glucose transporters GLUT1 or GLUT3 because SGLT1 has la lower Michaelis Menten K_m value and is able to accumulate intracellular glucose. When the extracellular cerebral glucose concentration is increased after prolonged ischemia or after TBI, up-regulated SGLT1 expression in neurons may be harmful. It may promote neuronal destruction because electrogenic sodium-D-glucose cotransport may lead to depolarization of neurons and increase neuronal activity by reducing the threshold for activation. On the other hand, SGLT1-mediated glucose transport into glial cells and/or neurons may reduce interstitial glucose concentration and thereby blunt edema formation (Yamazaki et al. 2012). In RS1^−/− mice post-traumatic SGLT1 expression levels are significantly lower compared to wild-type, whereas total brain glucose content in RS1^−/− mice was not different. The data suggest that RS1 dependent change in SGLT1 expression does not reduce the uptake of glucose from the blood stream but rather changes the redistribution of glucose in the brain.

Studying the role of SGLT1 and its regulation by RS1 after TBI we were faced with technical limitations which prompted us to determined changes of SGLT1 mRNA rather than protein as has been performed in studies concerning the role of SGLT1 during brain ischemia induced by MCAO. In the MCAO studies changes of SGLT1 protein were analyzed in Western blots using an antibody supplied by Millipore (Yamazaki et al. 2012, 2015). However, in our hands the commercial antibodies supplied by Merck Millipore (07-1417, Lot #2730935), Abcam (ab14686), and Santa Cruz Biotechnology (M-19, sc-20582) and also our own custom made antibody against mouse SGLT1 (Gorboulev et al. 2012) did not show specific immunocytochemical reaction with the low expressed SGLT1 protein in brain using the SGLT1^−/− mice as controls (Madunic et al. 2017). Actually, some immunocytochemical reactions observed in brain turned out to represent cross-reactivity because they were removed after preabsorption of the antibody with the antigenic peptides but remained in SGLT1^−/− mice. Measuring SGLT1 mRNA abundance after TBI we ensured detection of SGLT1 specific signals although the abundance of SGLT1 mRNA in brain is four orders of magnitude lower compared to small intestine where SGLT1 is the most abundant transport protein in the brush-border membrane (Wisniewski et al. 2015). Measuring mRNA abundance, we could relate the observed alterations to changes in transcription and/or mRNA stability. On the other hand, we were faced with the disadvantage that the mRNA levels may not show a one-to-one correlation to the expression of protein. However, also total cellular protein may not correlate with transporter abundance in the plasma membrane which in turn may not correlate with functional active protein.

The observation that SGLT1 mRNA abundance increased 1 day after CCI and that infarct volume and edema in the injured brain region and motoric impairment were decreased in parallel suggest pathophysiological impact of the up-regulation of SGLT1. The change of SGLT1 mRNA abundance observed in wild-type mice cannot be induced by a change in transcription of RS1 because RS1 mRNA abundance was increased 3 days after CCI. A pathophysiological role of the up-regulation of SGLT1 during CCI is not self-evident considering that in brain the mRNA concentration of SGLT1 is 1000 times smaller compared to GLUT1. However, it has to be considered that in contrast with GLUT1, SGLT1 is able to concentrate D-glucose in cells, that the turnover number of SGLT1-mediated, secondary active glucose transport may be higher compared to GLUT1, and that the membrane potential is decreased upon SGLT1 mediated sodium-D-glucose cotransport. Taken together the data suggest that SGLT1 could be a target for therapy during the early phase after brain trauma. Application of a specific SGLT1 inhibitor (Dobbins et al. 2015) or an inhibitor addressing SGLT1 plus SGLT2 (Zambrowicz et al. 2012) which could decrease elevated blood glucose during TBI in parallel may be effective, however, also the regulation of SGLT1 by RS1 may be addressed.

The finding that SGLT1 mRNA abundance after TBI was decreased in RS1^−/− mice was not expected because RS1 has been described to down-regulate SGLT1 expression. Previous data showed that RS1 which was localized in the cytosol, at the plasma membrane, at the Golgi, and within the nucleus (Kroiss et al. 2006), down-regulates SGLT1 on the transcriptional and post-translational levels (Korn et al. 2001; Veyhl et al. 2006; Chintalapati et al. 2016; Veyhl-Wichmann et al. 2016). In LLC-PK₁ cells it has been shown that confluence-dependent migration of RS1 into the nucleus is steered by a nuclear location signal (Filatova et al. 2009) and that removal of RS1 up-regulates SGLT1 transcription (Korn et al. 2001). The post-translational regulation of SGLT1 by RS1 was mainly characterized in oocytes of Xenopus leavis in which human SGLT1 was expressed and effects of injected RS1 fragments were determined. In this system, it was shown that a N-terminal, highly phosphorylated domain of RS1, termed RS1-Reg, blocks the release of SGLT1 containing vesicles, and that this hRS1-Reg-mediated blockage was prevented at high intracellular glucose concentrations (Veyhl-Wichmann et al. 2016). Evidence was provided that this regulation is responsible for short-term up-regulation of SGLT1 in small intestine after ingestion of glucose-rich food.

Our finding that SGLT1 mRNA abundance in brain was similar in RS1^−/− mice and wild-type mice whereas it was increased in small intestine and decreased in kidney indicates tissue specific RS1-mediated regulation of SGLT1 transcription and/or of mRNA stability. Our data show that the down-regulation of SGLT1 transcription and/or mRNA stability after TBI in RS1^−/− mice is beneficial for the clinical outcome, however, the effect of RS1 removal on SGLT1 transcription and/or mRNA stability may be counteracted by the loss of RS1-mediated post-translational down-regulation of SGLT1 protein in the plasma membrane. Thus, more pronounced beneficial effects may be obtained when the regulation of SGLT1 transcription and/or mRNA stability by RS1 and the post-translational regulation of SGLT1 by RS1 would be addressed separately. Provided glucose-induced relieve of RS1-mediated down-regulation is involved in glucose-dependent post-translational up-regulation of SGLT1 protein in the plasma membrane of neurons as has been shown in vitro and in small intestine, compounds blocking this glucose-induced up-regulation should be beneficial for the early treatment of TBI. We showed that a RS1-Reg mutant, in which serine in the Gln-Ser-Pro motif was replaced by glutamine, or the tripeptide Gln-Glu-Pro prevent the glucose-induced relieve of RS1-Reg-mediated down-regulation of SGLT1 in oocytes resulting in down-regulation of SGLT1 protein in the presence of high glucose concentrations (Veyhl-Wichmann et al. 2016) and unpublished data of U. Schäfer, M. Veyhl-Wichmann, and H. Koepsell.

Taken together, the present study shows that acute injury to the brain increases SGLT1 mRNA expression which is decreased after removal of the SGLT1 regulator RS1. These changes were accompanied by a reduced lesion volume and a mitigated motoric impairment in the early phase after insult indicating a pathophysiological role of SGLT1 during TBI. Inhibition of SGLT1 activity or down-regulation of SGLT1 with compounds derived from RS1 may be used for the early treatment of TBI to improve recovery and delay secondary brain damage.

Acknowledgments and conflict of interest disclosure

The authors thank Dana Pieter and Tobias Hirnet for their excellent technical assistance. This work was financially supported by the German Research Foundation (DFG TH1430/3-1). All authors declare that they have no conflict of interest.

All experiments were conducted in compliance with the ARRIVE guidelines.

Author contributions

All authors made a substantial contribution to the concept and design, acquisition of data or analysis and interpretation of data, drafted the article or revised it critically for important intellectual content, and approved the version to be published.

References

Bartnik-Olson B. L., Harris N. G., Shijo K. and Sutton R. L. (2013) Insights into the metabolic response to traumatic brain injury as revealed by (13)C NMR spectroscopy. Front. Neuro. 5, 8.
Google Scholar
Bergsneider M., Hovda D. A., Shalmon E. et al. (1997) Cerebral hyperglycolysis following severe traumatic brain injury in humans: a positron emission tomography study. J. Neurosurg. 86, 241–251.
10.3171/jns.1997.86.2.0241
CAS PubMed Web of Science® Google Scholar
Chen S., Pickard J. D. and Harris N. G. (2003) Time course of cellular pathology after controlled cortical impact injury. Exp. Neurol. 182, 87–102.
10.1016/S0014-4886(03)00002-5
CAS PubMed Web of Science® Google Scholar
Chintalapati C., Keller T., Mueller T. D. et al. (2016) Protein RS1 (RSC1A1) downregulates the exocytotic pathway of glucose transporter SGLT1 at Low intracellular glucose via Inhibition of ornithine decarboxylase. Mol. Pharmacol. 90, 508–521.
10.1124/mol.116.104521
CAS PubMed Web of Science® Google Scholar
Cook A. M., Peppard A. and Magnuson B. (2008) Nutrition considerations in traumatic brain injury. Nut. Clin. Prac. 23, 608–620.
10.1177/0884533608326060
PubMed Web of Science® Google Scholar
Cornford E. M., Hyman S., Cornford M. E. and Caron M. J. (1996) Glut1 glucose transporter activity in human brain injury. J. Neurotrauma 13, 523–536.
10.1089/neu.1996.13.523
CAS PubMed Web of Science® Google Scholar
De Salles A. A., Muizelaar J. P. and Young H. F. (1987) Hyperglycemia, cerebrospinal fluid lactic acidosis, and cerebral blood flow in severely head-injured patients. Neurosurgery 21, 45–50.
10.1227/00006123-198707000-00009
PubMed Web of Science® Google Scholar
Dietrich W. D., Alonso O. and Busto R. (1993) Moderate hyperglycemia worsens acute blood-brain barrier injury after forebrain ischemia in rats. Stroke 24, 111–116.
10.1161/01.STR.24.1.111
CAS PubMed Web of Science® Google Scholar
Dobbins R. L., Greenway F. L., Chen L., Liu Y., Breed S. L., Andrews S. M., Wald J. A., Walker A. and Smith C. D. (2015) Selective sodium-dependent glucose transporter 1 inhibitors block glucose absorption and impair glucose-dependent insulinotropic peptide release. Am. J. Physiol. Gastrointest. Liver Physiol. 308, G946–G954.
10.1152/ajpgi.00286.2014
CAS PubMed Web of Science® Google Scholar
Elfeber K., Kohler A., Lutzenburg M., Osswald C., Galla H. J., Witte O. W. and Koepsell H. (2004) Localization of the Na+-D-glucose cotransporter SGLT1 in the blood-brain barrier. Histochem. Cell Biol. 121, 201–207.
10.1007/s00418-004-0633-9
CAS PubMed Web of Science® Google Scholar
Faul F., Erdfelder E., Buchner A. and Lang A. G. (2009) Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behav. Res. Methods 41, 1149–1160.
10.3758/BRM.41.4.1149
PubMed Web of Science® Google Scholar
Ferreira J. M., Burnett A. L. and Rameau G. A. (2011) Activity-dependent regulation of surface glucose transporter-3. J. Neurosci. 31, 1991–1999.
10.1523/JNEUROSCI.1850-09.2011
CAS PubMed Web of Science® Google Scholar
Filatova A., Leyerer M., Gorboulev V., Chintalapati C., Reinders Y., Muller T. D., Srinivasan A., Hubner S. and Koepsell H. (2009) Novel shuttling domain in a regulator (RSC1A1) of transporter SGLT1 steers cell cycle-dependent nuclear location. Traffic 10, 1599–1618.
10.1111/j.1600-0854.2009.00982.x
CAS PubMed Web of Science® Google Scholar
Goodman J. C., Valadka A. B., Gopinath S. P., Uzura M. and Robertson C. S. (1999) Extracellular lactate and glucose alterations in the brain after head injury measured by microdialysis. Crit. Care Med. 27, 1965–1973.
10.1097/00003246-199909000-00041
CAS PubMed Web of Science® Google Scholar
Gorboulev V., Schurmann A., Vallon V. et al. (2012) Na(+)-D-glucose cotransporter SGLT1 is pivotal for intestinal glucose absorption and glucose-dependent incretin secretion. Diabetes 61, 187–196.
10.2337/db11-1029
CAS PubMed Web of Science® Google Scholar
Hamlin G. P., Cernak I., Wixey J. A. and Vink R. (2001) Increased expression of neuronal glucose transporter 3 but not glial glucose transporter 1 following severe diffuse traumatic brain injury in rats. J. Neurotrauma 18, 1011–1018.
10.1089/08977150152693700
CAS PubMed Web of Science® Google Scholar
Harada S., Fujita W. H., Shichi K. and Tokuyama S. (2009) The development of glucose intolerance after focal cerebral ischemia participates in subsequent neuronal damage. Brain Res. 1279, 174–181.
10.1016/j.brainres.2009.05.014
CAS PubMed Web of Science® Google Scholar
Harada S., Yamazaki Y., Nishioka H. and Tokuyama S. (2013) Neuroprotective effect through the cerebral sodium-glucose transporter on the development of ischemic damage in global ischemia. Brain Res. 1541, 61–68.
10.1016/j.brainres.2013.09.041
CAS PubMed Web of Science® Google Scholar
Ito D., Imai Y., Ohsawa K., Nakajima K., Fukuuchi Y. and Kohsaka S. (1998) Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res Mol Brain Res. 57(1), 1–9.
10.1016/S0169-328X(98)00040-0
CAS PubMed Google Scholar
Jeong H. K., Ji K., Min K. and Joe E. H. (2013) Brain inflammation and microglia: facts and misconceptions. Exper. Neurobiol. 22, 59–67.
10.5607/en.2013.22.2.59
PubMed Google Scholar
Jeremitsky E., Omert L. A., Dunham C. M., Wilberger J. and Rodriguez A. (2005) The impact of hyperglycemia on patients with severe brain injury. J. Trauma 58, 47–50.
10.1097/01.TA.0000135158.42242.B1
CAS PubMed Web of Science® Google Scholar
Koehler M. R., Lambotte S., Koepsell H. and Schmid M. (1996) Assignment of the membrane-associated modulator of the Na(+)-sugar cotransport systems gene RSC1A1 to human chromosome 1p36.1 by in situ hybridization. Cytogenet. Cell Genet. 73, 341–342.
10.1159/000134371
CAS PubMed Google Scholar
Koepsell H. (2017) The Na+-D-glucose cotransporters SGLT1 and SGLT2 are targets for the treatment of diabetes and cancer. Pharmacol. Ther. 170, 148–165.
10.1016/j.pharmthera.2016.10.017
CAS PubMed Web of Science® Google Scholar
Korn T., Kuhlkamp T., Track C., Schatz I., Baumgarten K., Gorboulev V. and Koepsell H. (2001) The plasma membrane-associated protein RS1 decreases transcription of the transporter SGLT1 in confluent LLC-PK1 cells. J. Biol. Chem. 276, 45330–45340.
10.1074/jbc.M105975200
CAS PubMed Web of Science® Google Scholar
Kroiss M., Leyerer M., Gorboulev V., Kuhlkamp T., Kipp H. and Koepsell H. (2006) Transporter regulator RS1 (RSC1A1) coats the trans-Golgi network and migrates into the nucleus. Am. J. Physiol. Renal Physiol. 291, F1201–F1212.
10.1152/ajprenal.00067.2006
CAS PubMed Web of Science® Google Scholar
Lam A. M., Winn H. R., Cullen B. F. and Sundling N. (1991) Hyperglycemia and neurological outcome in patients with head injury. J. Neurosurg. 75, 545–551.
10.3171/jns.1991.75.4.0545
CAS PubMed Web of Science® Google Scholar
Lambotte S., Veyhl M., Kohler M., Morrison-Shetlar A. I., Kinne R. K., Schmid M. and Koepsell H. (1996) The human gene of a protein that modifies Na(+)-D-glucose co-transport. DNA Cell Biol. 15, 769–777.
10.1089/dna.1996.15.769
CAS PubMed Web of Science® Google Scholar
Lee W. H. and Bondy C. A. (1993) Ischemic injury induces brain glucose transporter gene expression. Endocrinology 133, 2540–2544.
10.1210/endo.133.6.8243275
CAS PubMed Web of Science® Google Scholar
Lewen A., Matz P. and Chan P. H. (2000) Free radical pathways in CNS injury. J. Neurotrauma 17, 871–890.
10.1089/neu.2000.17.871
CAS PubMed Web of Science® Google Scholar
Li P. A., Liu G. J., He Q. P., Floyd R. A. and Siesjo B. K. (1999) Production of hydroxyl free radical by brain tissues in hyperglycemic rats subjected to transient forebrain ischemia. Free Radic. Biol. Med. 27, 1033–1040.
10.1016/S0891-5849(99)00152-5
CAS PubMed Web of Science® Google Scholar
Liu-DeRyke X., Collingridge D. S., Orme J., Roller D., Zurasky J. and Rhoney D. H. (2009) Clinical impact of early hyperglycemia during acute phase of traumatic brain injury. Neurocrit. Care 11, 151–157.
10.1007/s12028-009-9228-6
PubMed Web of Science® Google Scholar
Loane D. J. and Byrnes K. R. (2010) Role of microglia in neurotrauma. Neurotherapeutics 7, 366–377.
10.1016/j.nurt.2010.07.002
CAS PubMed Web of Science® Google Scholar
Luh C., Gierth K., Timaru-Kast R., Engelhard K., Werner C. and Thal S. C. (2011) Influence of a brief episode of anesthesia during the induction of experimental brain trauma on secondary brain damage and inflammation. PLoS ONE 6, e19948.
10.1371/journal.pone.0019948
CAS PubMed Web of Science® Google Scholar
Madunic I. V., Breljak D., Karaica D., Koepsell H. and Sabolic I. (2017) Expression profiling and immunolocalization of Na+-D-glucose-cotransporter 1 in mice employing knockout mice as specificity control indicate novel locations and differences between mice and rats. Pflugers Arch. 469, 1545–1565
10.1007/s00424-017-2056-1
CAS PubMed Web of Science® Google Scholar
Magistretti P. J. and Pellerin L. (1999) Astrocytes couple synaptic activity to glucose utilization in the brain. News Physiol. Sci. 14, 177–182.
CAS PubMed Web of Science® Google Scholar
Maher F., Vannucci S. J. and Simpson I. A. (1994) Glucose transporter proteins in brain. FASEB J. 8, 1003–1011.
10.1096/fasebj.8.13.7926364
CAS PubMed Web of Science® Google Scholar
Osswald C., Baumgarten K., Stumpel F. et al. (2005) Mice without the regulator gene Rsc1A1 exhibit increased Na+-D-glucose cotransport in small intestine and develop obesity. Mol. Cell. Biol. 25, 78–87.
10.1128/MCB.25.1.78-87.2005
CAS PubMed Web of Science® Google Scholar
Otori T., Friedland J. C., Sinson G., McIntosh T. K., Raghupathi R. and Welsh F. A. (2004) Traumatic brain injury elevates glycogen and induces tolerance to ischemia in rat brain. J. Neurotrauma 21, 707–718.
10.1089/0897715041269623
PubMed Web of Science® Google Scholar
Passonneau J. V. and Lauderdale V. R. (1974) A comparison of three methods of glycogen measurement in tissues. Anal. Biochem. 60, 405–412.
10.1016/0003-2697(74)90248-6
CAS PubMed Web of Science® Google Scholar
Pasternak J. J., McGregor D. G., Schroeder D. R. et al. (2008) Hyperglycemia in patients undergoing cerebral aneurysm surgery: its association with long-term gross neurologic and neuropsychological function. Mayo Clin. Proc. 83, 406–417.
10.4065/83.4.406
CAS PubMed Web of Science® Google Scholar
Poppe R., Karbach U., Gambaryan S., Wiesinger H., Lutzenburg M., Kraemer M., Witte O. W. and Koepsell H. (1997) Expression of the Na+-D-glucose cotransporter SGLT1 in neurons. J. Neurochem. 69, 84–94.
10.1046/j.1471-4159.1997.69010084.x
CAS PubMed Web of Science® Google Scholar
Roth T. L., Nayak D., Atanasijevic T., Koretsky A. P., Latour L. L. and McGavern D. B. (2014) Transcranial amelioration of inflammation and cell death after brain injury. Nature 505, 223–228.
10.1038/nature12808
CAS PubMed Web of Science® Google Scholar
Rovlias A. and Kotsou S. (2000) The influence of hyperglycemia on neurological outcome in patients with severe head injury. Neurosurgery 46, 335–342.
10.1097/00006123-200002000-00015
CAS PubMed Web of Science® Google Scholar
Scheepers A., Joost H. G. and Schurmann A. (2004) The glucose transporter families SGLT and GLUT: molecular basis of normal and aberrant function. JPEN. J. Parenter. Enteral. Nutr. 28, 364–371.
10.1177/0148607104028005364
CAS PubMed Web of Science® Google Scholar
Sebastiani A., Hirnet T., Jahn-Eimermacher A. and Thal S. C. (2017) Comparison of speed-vacuum method and heat-drying method to measure brain water content of small brain samples. J. Neurosci. Methods 276, 73–78.
10.1016/j.jneumeth.2016.11.012
PubMed Web of Science® Google Scholar
Thal S. C., Wyschkon S., Pieter D., Engelhard K. and Werner C. (2008) Selection of endogenous control genes for normalization of gene expression analysis after experimental brain trauma in mice. J. Neurotrauma 25, 785–794.
10.1089/neu.2007.0497
PubMed Web of Science® Google Scholar
Thal S. C., Luh C., Schaible E. V., Timaru-Kast R., Hedrich J., Luhmann H. J., Engelhard K. and Zehendner C. M. (2012) Volatile anesthetics influence blood-brain barrier integrity by modulation of tight junction protein expression in traumatic brain injury. PLoS ONE 7, e50752.
10.1371/journal.pone.0050752
CAS PubMed Web of Science® Google Scholar
Thal S. C., Schaible E. V., Neuhaus W., Scheffer D., Brandstetter M., Engelhard K., Wunder C. and Forster C. Y. (2013) Inhibition of proteasomal glucocorticoid receptor degradation restores dexamethasone-mediated stabilization of the blood-brain barrier after traumatic brain injury. Crit. Care Med. 41, 1305–1315.
10.1097/CCM.0b013e31827ca494
CAS PubMed Web of Science® Google Scholar
Thomas S., Prins M. L., Samii M. and Hovda D. A. (2000) Cerebral metabolic response to traumatic brain injury sustained early in development: a 2-deoxy-D-glucose autoradiographic study. J. Neurotrauma 17, 649–665.
10.1089/089771500415409
CAS PubMed Web of Science® Google Scholar
Urabe T., Hattori N., Nagamatsu S., Sawa H. and Mizuno Y. (1996) Expression of glucose transporters in rat brain following transient focal ischemic injury. J. Neurochem. 67, 265–271.
10.1046/j.1471-4159.1996.67010265.x
CAS PubMed Web of Science® Google Scholar
Vemula S., Roder K. E., Yang T., Bhat G. J., Thekkumkara T. J. and Abbruscato T. J. (2009) A functional role for sodium-dependent glucose transport across the blood-brain barrier during oxygen glucose deprivation. J. Pharmacol. Exp. Ther. 328, 487–495.
10.1124/jpet.108.146589
CAS PubMed Web of Science® Google Scholar
Veyhl M., Spangenberg J., Puschel B., Poppe R., Dekel C., Fritzsch G., Haase W. and Koepsell H. (1993) Cloning of a membrane-associated protein which modifies activity and properties of the Na(+)-D-glucose cotransporter. J. Biol. Chem. 268, 25041–25053.
CAS PubMed Web of Science® Google Scholar
Veyhl M., Keller T., Gorboulev V., Vernaleken A. and Koepsell H. (2006) RS1 (RSC1A1) regulates the exocytotic pathway of Na+-D-glucose cotransporter SGLT1. Am. J. Physiol. Renal Physiol. 291, F1213–F1223.
10.1152/ajprenal.00068.2006
CAS PubMed Web of Science® Google Scholar
Veyhl-Wichmann M., Friedrich A., Vernaleken A. et al. (2016) Phosphorylation of RS1 (RSC1A1) steers inhibition of different exocytotic pathways for glucose transporter SGLT1 and nucleoside transporter CNT1, and an RS1-derived peptide inhibits glucose absorption. Mol. Pharmacol. 89, 118–132.
10.1124/mol.115.101162
CAS PubMed Web of Science® Google Scholar
Widmer H., Abiko H., Faden A. I., James T. L. and Weinstein P. R. (1992) Effects of hyperglycemia on the time course of changes in energy metabolism and pH during global cerebral ischemia and reperfusion in rats: correlation of 1H and 31P NMR spectroscopy with fatty acid and excitatory amino acid levels. J. Cereb. Blood Flow Metab. 12, 456–468.
10.1038/jcbfm.1992.63
CAS PubMed Web of Science® Google Scholar
Wisniewski J. R., Friedrich A., Keller T., Mann M. and Koepsell H. (2015) The impact of high-fat diet on metabolism and immune defense in small intestine mucosa. J. Proteome Res. 14, 353–365.
10.1021/pr500833v
CAS PubMed Web of Science® Google Scholar
Wright E. M., Loo D. D. and Hirayama B. A. (2011) Biology of human sodium glucose transporters. Physiol. Rev. 91, 733–794.
10.1152/physrev.00055.2009
CAS PubMed Web of Science® Google Scholar
Yamazaki Y., Harada S. and Tokuyama S. (2012) Post-ischemic hyperglycemia exacerbates the development of cerebral ischemic neuronal damage through the cerebral sodium-glucose transporter. Brain Res. 1489, 113–120.
10.1016/j.brainres.2012.10.020
CAS PubMed Web of Science® Google Scholar
Yamazaki Y., Ogihara S., Harada S. and Tokuyama S. (2015) Activation of cerebral sodium-glucose transporter type 1 function mediated by post-ischemic hyperglycemia exacerbates the development of cerebral ischemia. Neuroscience 310, 674–685.
10.1016/j.neuroscience.2015.10.005
CAS PubMed Web of Science® Google Scholar
Yamazaki Y., Harada S., Wada T., Hagiwara T., Yoshida S. and Tokuyama S. (2017) Sodium influx through cerebral sodium-glucose transporter type 1 exacerbates the development of cerebral ischemic neuronal damage. Eur. J. Pharmacol. 799, 103–110.
10.1016/j.ejphar.2017.02.007
CAS PubMed Web of Science® Google Scholar
Yu A. S., Hirayama B. A., Timbol G. et al. (2010) Functional expression of SGLTs in rat brain. Am. J. Physiol. Cell Physiol. 299, C1277–C1284.
10.1152/ajpcell.00296.2010
CAS PubMed Web of Science® Google Scholar
Yu A. S., Hirayama B. A., Timbol G. et al. (2013) Regional distribution of SGLT activity in rat brain in vivo. Am. J. Physiol. Cell Physiol. 304, C240–C247.
10.1152/ajpcell.00317.2012
CAS PubMed Web of Science® Google Scholar
Zambrowicz B., Freiman J., Brown P. M. et al. (2012) LX4211, a dual SGLT1/SGLT2 inhibitor, improved glycemic control in patients with type 2 diabetes in a randomized, placebo-controlled trial. Clin. Pharmacol. Ther. 92, 158–169.
10.1038/clpt.2012.58
CAS PubMed Web of Science® Google Scholar
Zhang Z., Artelt M., Burnet M., Trautmann K. and Schluesener H. J. (2006) Early infiltration of CD8 + macrophages/microglia to lesions of rat traumatic brain injury. Neuroscience 141, 637–644.
10.1016/j.neuroscience.2006.04.027
CAS PubMed Web of Science® Google Scholar

Citing Literature

Volume147, Issue2

October 2018

Pages 190-203

RS1 (Rsc1A1) deficiency limits cerebral SGLT1 expression and delays brain damage after experimental traumatic brain injury