97
Journal of Alzheimer’s Disease 42 (2014) 97–101 DOI 10.3233/JAD-132498 IOS Press
Short Communication
Expression of the Glucose Transporter GLUT12 in Alzheimer’s Disease Patients Jonai Pujol-Gimeneza , Eva Martisovab , Alberto Perez-Mediavillab , Mar´ıa Pilar Lostaoa and Maria J. Ramirezb,c,∗ a Department
of Nutrition, Food Science and Physiology, University of Navarra, Pamplona, Spain of Cellular and Molecular Neuropharmacology, Division of Neurosciences, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain c Department of Pharmacology and Toxicology, University of Navarra, Pamplona, Spain b Department
Handling Associate Editor: Morty Mamelak
Accepted 19 March 2014
Abstract. Alzheimer’s disease (AD) might be conceptualized as a metabolic disease with progressive impairment of the brain’s capacity to utilize glucose. One of the last glucose transporters discovered is GLUT12. The aim of the present work was to investigate the expression of GLUT12 in frontal cortex from AD patients. Human samples from young control donors barely expressed GLUT12. The level of expression of GLUT12 was significantly higher in AD compare to aged controls. Expression of GLUT12 and Ox-42, a microglia marker, correlate in controls but not in AD. The implications of these findings in AD are discussed further. Keywords: Frontal cortex, glucose transport, microglia, western blot
Alzheimer’s disease (AD) is a neurodegenerative disorder that results in the progressive loss of memory and other cognitive functions. Aging is the primary risk factor for AD. Neuropathologically, AD brain is characterized by the accumulation of amyloid- (A) plaques and by neurofibrillary tangles caused by hyperphosphorylation of the tau protein. It is now widely acknowledged that AD might be conceptualized as a metabolic disease with progressive impairment of the brain’s capacity to utilize glucose and to respond to insulin [1, 2]. Glucose is an essential substrate for the maintenance of brain and neuronal activity. Glucose is taken up by ∗ Correspondence to: Maria J. Ramirez, Department of Pharmacology and Toxicology, University of Navarra, 31080 Pamplona, Spain. Tel.: +34 948 194700/Ext.: 2011; Fax: +34 948 194715; E-mail: [email protected].
brain cells through the facilitative glucose transporters GLUTs, which are expressed in all brain cell types [3]. Location, expression, and regulation of the different GLUT transporters are specific for each tissue and cellular type and are related to the cell metabolic needs. In many cases, the up- or down-regulation of the GLUT proteins is directly linked to the development of diseases [4]. One of the last discovered glucose transporters is GLUT12 [5]. GLUT12 cDNA was first isolated from the breast cancer cell line Mcf-7 and encodes for a 617 amino acids protein, which possess the structural features essential for sugar transport [4]. Immunoblotting assays have demonstrated GLUT12 expression in human skeletal muscle, adipose tissue, and small intestine, while in the brain only low levels of mRNA have been detected [5].
ISSN 1387-2877/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
98
J. Pujol-Gimenez et al. / GLUT12 and Alzheimer’s Disease
Research on GLUT12 has focused on its function on impaired insulin signaling pathologies [6]. It has been suggested that GLUT12 could complement the activity of other GLUTs in situations in which GLUT activity is decreased, as described in insulin resistance [7]. Interestingly, the expression of both GLUT1 and GLUT3, mainly responsible for brain glucose uptake in the brain [3], was shown to be reduced in the AD brain and directly correlated with hyperphosphorylation of tau [8]. It is hypothesized here that GLUT12 expression could be upregulated in AD to compensate for the loss of other GLUTs in the illness. The results show for the first time that GLUT12 is expressed in human brain and that is upregulated in AD patients. GLUT12 could be considered as a biomarker and/or potential target for the treatment of AD. Brain tissues were obtained from the Oxford Project to Investigate Memory and Ageing (OPTIMA, http:// www.medsci.ox.ac.uk/optima) and from the human brain tissue biobank “Biobanco Navarrabiomed”. Subjects were assessed annually for cognitive status using the Mini-Mental State Examination (MMSE). All subjects fulfilled CERAD criteria for the neuropathological diagnosis of AD and were staged at Braak V/VI. Controls did not have dementia or other neurological diseases, did not meet CERAD criteria for AD diagnosis, and were staged at Braak 0-II. Frontal (Brodmann Area, BA10) cortex was dissected free of meninges. All tissues used had a brain pH >6.1, condition used as an indicator of tissue quality in postmortem research [9]. A42 levels were determined using a commercially available high-sensitive ELISA kit (Wako Pure Chemical Industries, Tokyo, Japan) following manufacturer instructions. The expression of GLUT12 was determined using the rabbit anti-GLUT12 polyclonal antibody (1 : 500; bs-2540 R, Bioss, Woburn, MA, USA) in positive control tissues (Mcf-7 cell cultures, rat adipocytes, small intestine and muscle tissue homogenates) and in human cortical samples. Protein extracts (10–20 g) from control tissues were loaded onto an 8% bisacrylamide gels and separated by SDS-PAGE. BA10 homogenates (30 g) were loaded onto 10% Criterion XT BisTris Gels (Bio-Rad Laboratories, S.A., Madrid) and were run under reducing conditions. Separated proteins were electrophoretically transferred from the gels to nitrocellulose membranes (Hybond-ECL, Amersham Bioscience, UK). Ox-42 (rabbit polyclonal, 1 : 500, Thermo Scientific, Rockford, IL, USA) was also measured in the human samples. Preabsorption studies were carried out using the blocking peptide
of the GLUT12 antibody (1 : 50, GLUT12 peptide fragment of peptide, bs-2540P, Bioss, Woburn, MA, USA). Immunopositive bands were visualized using an enhanced chemiluminescence western blotting detection reagent (ECL; Amersham, Buckinghamshire, England). The optical density (O.D.) of the reactive bands, visible on x-ray film, was determined using densitometrically. -actin (mouse monoclonal, 1 : 10000, Sigma-Aldrich) was used as internal control of the loaded protein. Data were analyzed by SPSS for Windows, release 15.0. Normality was checked by Shapiro-Wilks’s test (p > 0.05). Differences were checked by Student’s t test. Intercorrelation between variables was investigated by Pearson’s or Spearman’s correlation coefficients, depending upon the normality of variables. The expression of GLUT12 was checked using a commercial antibody that provides a discrete band (68 KDa) in tissues in which the expression of this transporter has been demonstrated, such as MCF-7 cells, adipocytes, muscle, and intestine [5] (Fig. 1A). Noticeable bands were observed also at 110 and 225 KDa, which could correspond to oligomerization of this transporter, at similarity to other GLUTs [10]. All bands disappeared by pre-absorption of the antibody (Fig. 1B). Human frontal cortex samples from young control donors (age at death was 28 ± 5 years) barely expressed GLUT12, which is in accordance with previous results [5]. However, samples from aged controls showed measureable expression of GLUT12 (Fig. 2A). These results suggest that upregulation of the expression of GLUT12 might reflect an adaptive mechanism in response to physiological changes due to aging. Changes in the expression of GLUT12 in AD were next studied. The total number of cases analyzed was 12 controls (4 males/8 females) and 12 AD (5 males/7 females). There was a severe memory deficit in AD, and MMSE at death was 5 ± 1. Age at death was 75 ± 3 years for controls and 81 ± 2 for AD cases. There were no significant differences between controls and AD regarding postmortem delay (39 ± 5 h in controls versus 49 ± 6 h in AD) or brain pH (6.3 ± 0.2 in controls versus 6.4 ± 0.10 in AD). There were no significant correlations between age, postmortem delay, brain pH, or GLUT12 expression in either controls or AD. Interestingly, the level of expression of GLUT12 was significantly higher (∼40%) in AD compare to aged controls (Fig. 2B). The expression of the housekeeping protein -actin did not change in any case. So far, the main function described for GLUT12 is
J. Pujol-Gimenez et al. / GLUT12 and Alzheimer’s Disease
99
Fig. 1. A) GLUT12 expression in tissues identified as expressing GLUT12. B) Bands disappeared by pre-absorption.
Fig. 2. GLUT12 expression in frontal cortex of human brain samples. A) Expression in young vs. aged controls. Mcf-7 was used as positive control. B) Increased expression of GLUT12 in Alzheimer’s disease (AD) samples (upper panel). In each panel, a representative picture of western blot (upper panel) and quantification of data (lower panel) is shown. n = 12 in each group. Results are normalized to -actin and expressed as % optical density (O.D.) of controls. *p < 0.05 Student’s t-test.
100
J. Pujol-Gimenez et al. / GLUT12 and Alzheimer’s Disease
its activity as a secondary insulin-sensitive transporter [11]. The increase in GLUT12 expression in AD could be a compensatory mechanism to counter balance the impaired insulin response characteristic of the insulinresistance which develops in AD brains. Supporting this hypothesis, overexpression of GLUT12 in transgenic mice resulted in a higher expression of the transporter in the brain compared with other tissues, and improved whole-body insulin sensitivity [12]. The contribution of aging to this process has to be considered as well, as GLUT12 expression was already increased in aged compared to young controls. Previous works have showed that GLUT4 expression was not modified in AD [8] and since GLUT12 functional properties and regulation are very different to those depicted for GLUT4 [4], it could be argue that GLUT12 activity would be complementary to GLUT4. Nevertheless, GLUT12 could also be considered as an evolutionary ancestor retained as backup of GLUT4, as previously proposed [11]. Deposition of the A peptide to the cortical vessel walls produces cerebral amyloid angiopathy that could contribute to the hypoperfusion of cerebral tissue. In this regard, as expected, A42 levels were significantly increased in AD cases compared to controls (29.34 ± 2.33 pg/ml in controls; 37.85 ± 4.01 pg/ml in AD, p < 0.05). However, no correlation between A42 levels and GLUT12 expression was found. GLUT12 overexpression could be also related to the increase of lactate release through the activation of the anaerobic glycolytic metabolism. AD show a progressive impairment of the brain’s capacity to respond to insulin [1]. GLUT12 expression has been described in different cancer cell lines, where it seems to act as a key protein to keep the energy supply through the anaerobic glycolytic metabolism [13]. Defects in brain glucose transport, disrupted glycolysis, and/or impaired mitochondrial function are involved in the brain hypometabolism related to the cognitive decline in AD [14]. Interestingly, in AD brains the level of lactate is elevated in the cerebrospinal fluid [15]. Moreover, neuroblastoma cells exposed to high lactic acid levels increase the secretion of A42 [16]. Therefore, alternatively to its role in the insulin response, it could be hypothesized that GLUT12 over expression in AD could be related to glycolytic metabolism. Expression of GLUT12 and Ox-42, a microglia activation marker, correlate in controls (r = 0.667; p < 0.05, Spearman’s correlation coefficient). However, even though Ox-42 expression was significantly enhanced in AD over controls (4-fold increase, p < 0.01), the correlation between OX-42 and GLUT12 expression was
lost in AD. Therefore, it does not seem that GLUT12 location is restricted to microglia. In this work, some possible scenarios have been presented in which GLUT12 could play a role in AD. As a final comment, some limitations of the present study should be acknowledged: relative small number of cases (and only severe AD cases) and only one brain region. Even though a deeper knowledge of the functional properties and brain location of GLUT12 is needed to understand its role in AD, GLUT12 seems to be involved in AD and therefore, it could be considered, as other GLUTs [17, 18], a useful tool both as biomarker or therapeutic target to fight against AD. ACKNOWLEDGMENTS We thank A. Red´ın for technical assistance. This work has been supported by grants from FIS (10/01748), “Tu eliges, Tu decides” projects, “Fundaci´on Bot´ın” and PIUNA (University of Navarra). JPG was a recipient of a fellowship from “Asociaci´on de Amigos” (University of Navarra). Brain tissues were obtained from the Oxford Project to Investigate Memory and Ageing/Thomas Willis Oxford Brain Collection, part of the Brains for Dementia Research network. Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=2217). REFERENCES [1]
[2]
[3]
[4]
[5]
[6]
[7] [8]
de la Monte SM (2012) Brain insulin resistance and deficiency as therapeutic targets in Alzheimer’s disease. Curr Alzheimer Res 9, 35-66. De Felice FG (2013) Alzheimer’s disease and insulin resistance: Translating basic science into clinical applications. J Clin Invest 123, 531-539. Simpson IA, Carruthers A, Vannucci SJ (2007) Supply and demand in cerebral energy metabolism: The role of nutrient transporters. J Cereb Blood Flow Metab 27, 1766-1791. Pujol-Gim´enez J, Barrenetxe J, Gonz´alez-Muniesa P, Lostao MP (2013) The facilitative glucose transporter GLUT12: What do we know and what would we like to know? J Physiol Biochem 69, 325-333. Rogers S, Macheda ML, Docherty SE, Carty MD, Henderson MA, Soeller WC, Gibbs EM, James DE, Best JD (2002) Identification of a novel glucose transporter-like proteinGLUT-12. Am J Physiol Endocrinol Metab 282, E733-E738. Waller AP, Kohler K, Burns TA, Mudge MC, Belknap JK, Lacombe VA (2011) Naturally occurring compensated insulin resistance selectively alters glucose transporters in visceral and subcutaneous adipose tissues without change in AS160 activation. Biochim Biophys Acta 1812, 1098-1103. Uldry M, Thorens B (2004) The SLC2 family of facilitated hexose and polyol transporters. Pflugers Arch 447, 480-489. Liu Y, Liu F, Iqbal K, Grundke-Iqbal I, Gong CX (2008) Decreased glucose transporters correlate to abnormal
J. Pujol-Gimenez et al. / GLUT12 and Alzheimer’s Disease
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[10]
[11]
[12]
[13]
[14]
hyperphosphorylation of tau in Alzheimer disease. FEBS Lett 582, 359-364. Lewis DA (2002) The human brain revisited: Opportunities and challenges in postmortem studies of psychiatric disorders. Neuropsychopharmacol 26, 143-154. De Zutter JK, Levine KB, Deng D, Carruthers A (2013) Sequence determinants of GLUT1 oligomerization – analysisby homology-scanning mutagenesis. J Biol Chem 288, 20734-20744. Stuart CA, Howell ME, Zhang Y, Yin D (2009) Insulinstimulated translocation of glucose transporter (GLUT) 12 parallels that of GLUT4 in normal muscle. J Clin Endocrinol Metab 94, 3535-3542. Purcell SH, Aerni-Flessner LB, Willcockson AR, DiggsAndrews KA, Fisher SJ, Moley KH (2011) Improved insulin sensitivity by GLUT12 overexpression in mice. Diabetes 60, 1478-1482. Zawacka-Pankau J, Grinkevich VV, H¨unten S, Nikulenkov F, Gluch A, Li H, Enge M, Kel A, Selivanova G (2011) Inhibition of glycolytic enzymes mediated by pharmacologically activated p53: Targeting Warburg effect to fight cancer. J Biol Chem 286, 41600-41615. Cunnane S, Nugent S, Roy M, Courchesne-Loyer A, Croteau E, Tremblay S, Castellano A, Pifferi F, Bocti C, Paquet N,
[15]
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[17]
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Begdouri H, Bentourkia M, Turcotte E, Allard M, BarbergerGateau P, Fulop T, Rapoport SI (2011) Brain fuel metabolism, aging, and Alzheimer’sdisease. Nutrition 27, 3-20. Redjems-Bennani N, Jeandel C, Lefebvre E, Blain H, Vidailhet M, Gueant JL (1998) Abnormal substrate levels that depend upon mitochondrial function in cerebrospinal fluid from Alzheimer patients. Gerontology 44, 300304. Xiang Y, Xu G, Weigel-Van Aken KA (2010) Lactic acid induces aberrant amyloid precursor protein processing by promoting its interaction with endoplasmic reticulum chaperone proteins. PLoS One 5, e13820. Evans A, Bates V, Troy H, Hewitt S, Holbeck S, Chung YL, Phillips R, Stubbs M, Griffiths J, Airley R (2008) Glut-1 as a therapeutic target: Increased chemoresistance and HIF-1-independent link with cell turnover is revealed through COMPARE analysis and metabolomic studies. Cancer Chemother Pharmacol 61, 377-393. Liu P, Lu Y, Gao X, Liu R, Zhang-Negrerie D, Shi Y, Wang Y, Wang S, Gao Q (2013) Highly water-soluble platinum(II) complexes as GLUT substrates for targeted therapy: Improved anticancer efficacy and transporter-mediated cytotoxic properties. Chem Commun 49, 2421-2423.
Journal of Alzheimer’s Disease 42 (2014) 97–101 DOI 10.3233/JAD-132498 IOS Press
Short Communication
Expression of the Glucose Transporter GLUT12 in Alzheimer’s Disease Patients Jonai Pujol-Gimeneza , Eva Martisovab , Alberto Perez-Mediavillab , Mar´ıa Pilar Lostaoa and Maria J. Ramirezb,c,∗ a Department
of Nutrition, Food Science and Physiology, University of Navarra, Pamplona, Spain of Cellular and Molecular Neuropharmacology, Division of Neurosciences, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain c Department of Pharmacology and Toxicology, University of Navarra, Pamplona, Spain b Department
Handling Associate Editor: Morty Mamelak
Accepted 19 March 2014
Abstract. Alzheimer’s disease (AD) might be conceptualized as a metabolic disease with progressive impairment of the brain’s capacity to utilize glucose. One of the last glucose transporters discovered is GLUT12. The aim of the present work was to investigate the expression of GLUT12 in frontal cortex from AD patients. Human samples from young control donors barely expressed GLUT12. The level of expression of GLUT12 was significantly higher in AD compare to aged controls. Expression of GLUT12 and Ox-42, a microglia marker, correlate in controls but not in AD. The implications of these findings in AD are discussed further. Keywords: Frontal cortex, glucose transport, microglia, western blot
Alzheimer’s disease (AD) is a neurodegenerative disorder that results in the progressive loss of memory and other cognitive functions. Aging is the primary risk factor for AD. Neuropathologically, AD brain is characterized by the accumulation of amyloid- (A) plaques and by neurofibrillary tangles caused by hyperphosphorylation of the tau protein. It is now widely acknowledged that AD might be conceptualized as a metabolic disease with progressive impairment of the brain’s capacity to utilize glucose and to respond to insulin [1, 2]. Glucose is an essential substrate for the maintenance of brain and neuronal activity. Glucose is taken up by ∗ Correspondence to: Maria J. Ramirez, Department of Pharmacology and Toxicology, University of Navarra, 31080 Pamplona, Spain. Tel.: +34 948 194700/Ext.: 2011; Fax: +34 948 194715; E-mail: [email protected].
brain cells through the facilitative glucose transporters GLUTs, which are expressed in all brain cell types [3]. Location, expression, and regulation of the different GLUT transporters are specific for each tissue and cellular type and are related to the cell metabolic needs. In many cases, the up- or down-regulation of the GLUT proteins is directly linked to the development of diseases [4]. One of the last discovered glucose transporters is GLUT12 [5]. GLUT12 cDNA was first isolated from the breast cancer cell line Mcf-7 and encodes for a 617 amino acids protein, which possess the structural features essential for sugar transport [4]. Immunoblotting assays have demonstrated GLUT12 expression in human skeletal muscle, adipose tissue, and small intestine, while in the brain only low levels of mRNA have been detected [5].
ISSN 1387-2877/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
98
J. Pujol-Gimenez et al. / GLUT12 and Alzheimer’s Disease
Research on GLUT12 has focused on its function on impaired insulin signaling pathologies [6]. It has been suggested that GLUT12 could complement the activity of other GLUTs in situations in which GLUT activity is decreased, as described in insulin resistance [7]. Interestingly, the expression of both GLUT1 and GLUT3, mainly responsible for brain glucose uptake in the brain [3], was shown to be reduced in the AD brain and directly correlated with hyperphosphorylation of tau [8]. It is hypothesized here that GLUT12 expression could be upregulated in AD to compensate for the loss of other GLUTs in the illness. The results show for the first time that GLUT12 is expressed in human brain and that is upregulated in AD patients. GLUT12 could be considered as a biomarker and/or potential target for the treatment of AD. Brain tissues were obtained from the Oxford Project to Investigate Memory and Ageing (OPTIMA, http:// www.medsci.ox.ac.uk/optima) and from the human brain tissue biobank “Biobanco Navarrabiomed”. Subjects were assessed annually for cognitive status using the Mini-Mental State Examination (MMSE). All subjects fulfilled CERAD criteria for the neuropathological diagnosis of AD and were staged at Braak V/VI. Controls did not have dementia or other neurological diseases, did not meet CERAD criteria for AD diagnosis, and were staged at Braak 0-II. Frontal (Brodmann Area, BA10) cortex was dissected free of meninges. All tissues used had a brain pH >6.1, condition used as an indicator of tissue quality in postmortem research [9]. A42 levels were determined using a commercially available high-sensitive ELISA kit (Wako Pure Chemical Industries, Tokyo, Japan) following manufacturer instructions. The expression of GLUT12 was determined using the rabbit anti-GLUT12 polyclonal antibody (1 : 500; bs-2540 R, Bioss, Woburn, MA, USA) in positive control tissues (Mcf-7 cell cultures, rat adipocytes, small intestine and muscle tissue homogenates) and in human cortical samples. Protein extracts (10–20 g) from control tissues were loaded onto an 8% bisacrylamide gels and separated by SDS-PAGE. BA10 homogenates (30 g) were loaded onto 10% Criterion XT BisTris Gels (Bio-Rad Laboratories, S.A., Madrid) and were run under reducing conditions. Separated proteins were electrophoretically transferred from the gels to nitrocellulose membranes (Hybond-ECL, Amersham Bioscience, UK). Ox-42 (rabbit polyclonal, 1 : 500, Thermo Scientific, Rockford, IL, USA) was also measured in the human samples. Preabsorption studies were carried out using the blocking peptide
of the GLUT12 antibody (1 : 50, GLUT12 peptide fragment of peptide, bs-2540P, Bioss, Woburn, MA, USA). Immunopositive bands were visualized using an enhanced chemiluminescence western blotting detection reagent (ECL; Amersham, Buckinghamshire, England). The optical density (O.D.) of the reactive bands, visible on x-ray film, was determined using densitometrically. -actin (mouse monoclonal, 1 : 10000, Sigma-Aldrich) was used as internal control of the loaded protein. Data were analyzed by SPSS for Windows, release 15.0. Normality was checked by Shapiro-Wilks’s test (p > 0.05). Differences were checked by Student’s t test. Intercorrelation between variables was investigated by Pearson’s or Spearman’s correlation coefficients, depending upon the normality of variables. The expression of GLUT12 was checked using a commercial antibody that provides a discrete band (68 KDa) in tissues in which the expression of this transporter has been demonstrated, such as MCF-7 cells, adipocytes, muscle, and intestine [5] (Fig. 1A). Noticeable bands were observed also at 110 and 225 KDa, which could correspond to oligomerization of this transporter, at similarity to other GLUTs [10]. All bands disappeared by pre-absorption of the antibody (Fig. 1B). Human frontal cortex samples from young control donors (age at death was 28 ± 5 years) barely expressed GLUT12, which is in accordance with previous results [5]. However, samples from aged controls showed measureable expression of GLUT12 (Fig. 2A). These results suggest that upregulation of the expression of GLUT12 might reflect an adaptive mechanism in response to physiological changes due to aging. Changes in the expression of GLUT12 in AD were next studied. The total number of cases analyzed was 12 controls (4 males/8 females) and 12 AD (5 males/7 females). There was a severe memory deficit in AD, and MMSE at death was 5 ± 1. Age at death was 75 ± 3 years for controls and 81 ± 2 for AD cases. There were no significant differences between controls and AD regarding postmortem delay (39 ± 5 h in controls versus 49 ± 6 h in AD) or brain pH (6.3 ± 0.2 in controls versus 6.4 ± 0.10 in AD). There were no significant correlations between age, postmortem delay, brain pH, or GLUT12 expression in either controls or AD. Interestingly, the level of expression of GLUT12 was significantly higher (∼40%) in AD compare to aged controls (Fig. 2B). The expression of the housekeeping protein -actin did not change in any case. So far, the main function described for GLUT12 is
J. Pujol-Gimenez et al. / GLUT12 and Alzheimer’s Disease
99
Fig. 1. A) GLUT12 expression in tissues identified as expressing GLUT12. B) Bands disappeared by pre-absorption.
Fig. 2. GLUT12 expression in frontal cortex of human brain samples. A) Expression in young vs. aged controls. Mcf-7 was used as positive control. B) Increased expression of GLUT12 in Alzheimer’s disease (AD) samples (upper panel). In each panel, a representative picture of western blot (upper panel) and quantification of data (lower panel) is shown. n = 12 in each group. Results are normalized to -actin and expressed as % optical density (O.D.) of controls. *p < 0.05 Student’s t-test.
100
J. Pujol-Gimenez et al. / GLUT12 and Alzheimer’s Disease
its activity as a secondary insulin-sensitive transporter [11]. The increase in GLUT12 expression in AD could be a compensatory mechanism to counter balance the impaired insulin response characteristic of the insulinresistance which develops in AD brains. Supporting this hypothesis, overexpression of GLUT12 in transgenic mice resulted in a higher expression of the transporter in the brain compared with other tissues, and improved whole-body insulin sensitivity [12]. The contribution of aging to this process has to be considered as well, as GLUT12 expression was already increased in aged compared to young controls. Previous works have showed that GLUT4 expression was not modified in AD [8] and since GLUT12 functional properties and regulation are very different to those depicted for GLUT4 [4], it could be argue that GLUT12 activity would be complementary to GLUT4. Nevertheless, GLUT12 could also be considered as an evolutionary ancestor retained as backup of GLUT4, as previously proposed [11]. Deposition of the A peptide to the cortical vessel walls produces cerebral amyloid angiopathy that could contribute to the hypoperfusion of cerebral tissue. In this regard, as expected, A42 levels were significantly increased in AD cases compared to controls (29.34 ± 2.33 pg/ml in controls; 37.85 ± 4.01 pg/ml in AD, p < 0.05). However, no correlation between A42 levels and GLUT12 expression was found. GLUT12 overexpression could be also related to the increase of lactate release through the activation of the anaerobic glycolytic metabolism. AD show a progressive impairment of the brain’s capacity to respond to insulin [1]. GLUT12 expression has been described in different cancer cell lines, where it seems to act as a key protein to keep the energy supply through the anaerobic glycolytic metabolism [13]. Defects in brain glucose transport, disrupted glycolysis, and/or impaired mitochondrial function are involved in the brain hypometabolism related to the cognitive decline in AD [14]. Interestingly, in AD brains the level of lactate is elevated in the cerebrospinal fluid [15]. Moreover, neuroblastoma cells exposed to high lactic acid levels increase the secretion of A42 [16]. Therefore, alternatively to its role in the insulin response, it could be hypothesized that GLUT12 over expression in AD could be related to glycolytic metabolism. Expression of GLUT12 and Ox-42, a microglia activation marker, correlate in controls (r = 0.667; p < 0.05, Spearman’s correlation coefficient). However, even though Ox-42 expression was significantly enhanced in AD over controls (4-fold increase, p < 0.01), the correlation between OX-42 and GLUT12 expression was
lost in AD. Therefore, it does not seem that GLUT12 location is restricted to microglia. In this work, some possible scenarios have been presented in which GLUT12 could play a role in AD. As a final comment, some limitations of the present study should be acknowledged: relative small number of cases (and only severe AD cases) and only one brain region. Even though a deeper knowledge of the functional properties and brain location of GLUT12 is needed to understand its role in AD, GLUT12 seems to be involved in AD and therefore, it could be considered, as other GLUTs [17, 18], a useful tool both as biomarker or therapeutic target to fight against AD. ACKNOWLEDGMENTS We thank A. Red´ın for technical assistance. This work has been supported by grants from FIS (10/01748), “Tu eliges, Tu decides” projects, “Fundaci´on Bot´ın” and PIUNA (University of Navarra). JPG was a recipient of a fellowship from “Asociaci´on de Amigos” (University of Navarra). Brain tissues were obtained from the Oxford Project to Investigate Memory and Ageing/Thomas Willis Oxford Brain Collection, part of the Brains for Dementia Research network. Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=2217). REFERENCES [1]
[2]
[3]
[4]
[5]
[6]
[7] [8]
de la Monte SM (2012) Brain insulin resistance and deficiency as therapeutic targets in Alzheimer’s disease. Curr Alzheimer Res 9, 35-66. De Felice FG (2013) Alzheimer’s disease and insulin resistance: Translating basic science into clinical applications. J Clin Invest 123, 531-539. Simpson IA, Carruthers A, Vannucci SJ (2007) Supply and demand in cerebral energy metabolism: The role of nutrient transporters. J Cereb Blood Flow Metab 27, 1766-1791. Pujol-Gim´enez J, Barrenetxe J, Gonz´alez-Muniesa P, Lostao MP (2013) The facilitative glucose transporter GLUT12: What do we know and what would we like to know? J Physiol Biochem 69, 325-333. Rogers S, Macheda ML, Docherty SE, Carty MD, Henderson MA, Soeller WC, Gibbs EM, James DE, Best JD (2002) Identification of a novel glucose transporter-like proteinGLUT-12. Am J Physiol Endocrinol Metab 282, E733-E738. Waller AP, Kohler K, Burns TA, Mudge MC, Belknap JK, Lacombe VA (2011) Naturally occurring compensated insulin resistance selectively alters glucose transporters in visceral and subcutaneous adipose tissues without change in AS160 activation. Biochim Biophys Acta 1812, 1098-1103. Uldry M, Thorens B (2004) The SLC2 family of facilitated hexose and polyol transporters. Pflugers Arch 447, 480-489. Liu Y, Liu F, Iqbal K, Grundke-Iqbal I, Gong CX (2008) Decreased glucose transporters correlate to abnormal
J. Pujol-Gimenez et al. / GLUT12 and Alzheimer’s Disease
[9]
[10]
[11]
[12]
[13]
[14]
hyperphosphorylation of tau in Alzheimer disease. FEBS Lett 582, 359-364. Lewis DA (2002) The human brain revisited: Opportunities and challenges in postmortem studies of psychiatric disorders. Neuropsychopharmacol 26, 143-154. De Zutter JK, Levine KB, Deng D, Carruthers A (2013) Sequence determinants of GLUT1 oligomerization – analysisby homology-scanning mutagenesis. J Biol Chem 288, 20734-20744. Stuart CA, Howell ME, Zhang Y, Yin D (2009) Insulinstimulated translocation of glucose transporter (GLUT) 12 parallels that of GLUT4 in normal muscle. J Clin Endocrinol Metab 94, 3535-3542. Purcell SH, Aerni-Flessner LB, Willcockson AR, DiggsAndrews KA, Fisher SJ, Moley KH (2011) Improved insulin sensitivity by GLUT12 overexpression in mice. Diabetes 60, 1478-1482. Zawacka-Pankau J, Grinkevich VV, H¨unten S, Nikulenkov F, Gluch A, Li H, Enge M, Kel A, Selivanova G (2011) Inhibition of glycolytic enzymes mediated by pharmacologically activated p53: Targeting Warburg effect to fight cancer. J Biol Chem 286, 41600-41615. Cunnane S, Nugent S, Roy M, Courchesne-Loyer A, Croteau E, Tremblay S, Castellano A, Pifferi F, Bocti C, Paquet N,
[15]
[16]
[17]
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