brain research 1489 (2012) 113–120
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Post-ischemic hyperglycemia exacerbates the development of cerebral ischemic neuronal damage through the cerebral sodium-glucose transporter Yui Yamazaki, Shinichi Harada, Shogo Tokuyaman Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe 650-8586, Japan
art i cle i nfo
ab st rac t
Article history:
Post-ischemic hyperglycemia may be one of the triggers of ischemic neuronal damage.
Accepted 9 October 2012
However, the detailed mechanisms of this injury process are still unknown. Here, we
Available online 15 October 2012
focused on the involvement of the sodium-glucose transporter (SGLT), which transports
Keywords:
glucose together with Naþ ions, and generates inward currents while transporting glucose
Glucose transporter
into cells, resulting in depolarization and increased excitability. The aim of this study was
Cerebral ischemia
to determine the involvement of the SGLT in the development of cerebral ischemic stress-
Hyperglycemia
induced neuronal damage. Male ddY mice were subjected to 2 h of middle cerebral artery
SGLT
occlusion (MCAO). Fasting blood glucose (FBG) was measured using the glucose pilot.
Neuroprotection
Neuronal damage was estimated by histological and behavioral analyses. Phlorizin and glucose were administered by intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) injection. Administration of phlorizin (40, 120 or 200 mg/kg, i.p.) significantly and dosedependently suppressed the elevation of FBG and ischemic neuronal damage. In contrast, phlorizin (10 or 40 mg/mouse, i.c.v.) significantly and dose-dependently suppressed ischemic neuronal damage without reducing the elevation of FBG. Moreover, the development of neuronal damage was significantly and dose-dependently exacerbated following i.c.v. administration of glucose (10% or 25% (w/v)), and its exacerbation was suppressed by i.c.v. administration of phlorizin (40 mg/mouse). These results suggest that cerebral SGLT is activated by post-ischemic hyperglycemia and may be involved in the exacerbation of ischemic neuronal damage. & 2012 Elsevier B.V. All rights reserved.
1.
Introduction
Ischemic stroke has been identified as the third leading cause of death and disability worldwide, and involves focal neurologic deficits caused by a change in cerebral circulation (Shuaib et al., 2007). Many risk factors, including blood n
Corresponding author. Fax: þ81 78 974 4780. E-mail address:
[email protected] (S. Tokuyama).
0006-8993/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2012.10.020
pressure, diabetes, and hyperlipidemia, have been known to contribute to stroke onset (Kernan et al., 2005; Saito, 2012). In particular, diabetes and/or hyperglycemic conditions before development of cerebral ischemia induce more severe strokes (Fuentes et al., 2009; Harada et al., 2012). In addition, it has been clinically reported that the vast majority of patients
114
brain research 1489 (2012) 113–120
affected with acute cerebral ischemia have abnormal regulation of glucose metabolism (Matz et al., 2006). In contrast, regardless if a patient has a history of diabetes or not, hyperglycemia and/or glucose intolerance are reported to be induced after cerebral ischemia, which is termed postischemic hyperglycemia (Matz et al., 2006). We have previously reported that post-ischemic hyperglycemia develops in the acute phase of cerebral ischemia using focal cerebral ischemia model mice (Harada et al., 2009a, 2011). Moreover, cerebral ischemic neuronal damage has been reported to improve post-ischemic glucose intolerance in the presence of anti-diabetic drugs, such as insulin or metformin (Harada et al., 2009a, 2010). However, the mechanisms of post-ischemic hyperglycemia-induced exacerbation of ischemic neuronal damage are still unclear. Hence, we focused on the involvement of the sodium-glucose transporter (SGLT), which is a known glucose transporter. The SGLT family belongs to the solute carrier 5 A family and has six isoforms (Perez Lopez et al., 2010). The SGLT has been reported to transport glucose together with Naþ ions, and generate inward currents in the process of transporting glucose into cells, resulting in depolarization and increased excitability (Diez-Sampedro et al., 2003; Scheepers et al., 2004). In addition, the SGLT induces excitotoxicity-mediated neuronal death through Ca2þ influx into neurons via Naþ-dependent depolarization (PellegriniGiampietro et al., 1997). Furthermore, it is well known that neuronal death is caused by high glucose conditions, and is exacerbated by glucose-induced increases in oxidative stress such as reactive oxygen species (Costa et al., 2012). Glucose uptake in tissue or neurons also induces the production of energy and activates caspases such as caspase-3, which is a key enzyme involved in the induction of apoptosis (Benchoua et al., 2001). As identified above, it is possible that post-ischemic hyperglycemia may induce excessive activation of the SGLT family leading to depolarization-induced neuronal damage. Therefore, the aim of this study was to determine the involvement of the SGLT in the development of cerebral ischemic stressinduced neuronal damage.
2.
Results
2.1. Effect of phlorizin (i.p.) on the elevation of FBG after cerebral ischemic stress Fasting blood glucose levels (FBG) was significantly increased on day 1 after middle cerebral artery occlusion (MCAO) as compared with the sham group (Fig. 1). Phlorizin (40, 120 or 200 mg/kg) significantly suppressed the elevation of FBG in a dose-dependent manner on day 1 after MCAO as compared with the vehicle-treated group (Fig. 1). In contrast, phlorizin itself did not have any effect on FBG (Fig. 1).
2.2. Effect of phlorizin (i.p.) on the development of neuronal damage after cerebral ischemic stress Enhancement of infarct volume was dose-dependently suppressed on day 3 after MCAO in the phlorizin (40, 120 or 200 mg/kg)-treated group as compared with the vehicletreated group, and the effect of 200 mg/kg of phlorizin was significant (Fig. 2A and B). In addition, phlorizin (40, 120 or 200 mg/kg) improved the increase in neurological deficit score (NDS) similar to that of infarct volume (Fig. 2C).
2.3. Effect of phlorizin (i.c.v.) on the elevation of FBG after cerebral ischemic stress Phlorizin (10 or 40 mg/mouse) did not affect the elevation of FBG on day 1 after MCAO (Fig. 3).
2.4. Effect of phlorizin (i.c.v.) on the development of neuronal damage after cerebral ischemic stress The enhancement of infarct volume was suppressed on day 3 after MCAO in the phlorizin (10 or 40 mg/mouse)-treated group in a dose-dependent manner as compared with the vehicletreated group (Fig. 4A and B). Furthermore, the increase in NDS was also significantly and dose-dependently decreased in a similar manner to that of infarct volume (Fig. 4C).
Fig. 1 – Effect of phlorizin (i.p.) on the elevation of FBG after cerebral ischemic stress. Mice were treated with phlorizin (40, 120 or 200 mg/kg, i.p.) immediately after reperfusion. Effect of phlorizin on fasting blood glucose levels on day 1 after cerebral ischemia. Results are presented as the mean7S.E.M. nnPo0.01, #Po0.05. vehicle-treated sham group: n ¼12, 40 mg/kg phlorizin-treated sham group: n ¼6, 120 mg/kg phlorizin-treated sham group: n ¼ 4, 200 mg/kg phlorizin-treated sham group: n¼ 6, vehicle-treated MCAO group: n¼ 17, 40 mg/kg phlorizin-treated MCAO group: n ¼ 6, 120 mg/kg phlorizin-treated MCAO group: n¼ 7, 200 mg/kg phlorizin-treated MCAO group: n ¼ 12. veh: vehicle, PHZ: phlorizin.
brain research 1489 (2012) 113–120
115
Fig. 2 – Effect of phlorizin (i.p.) on the development of neuronal damage after cerebral ischemic stress. Representative photographs of TTC staining on day 3 after MCAO. (A) Quantitative analysis of infarct volume. (B) Results are presented as the mean7S.E.M. nnPo0.01, vehicle-treated MCAO group: n¼ 17, 40 mg/kg phlorizin-treated MCAO group: n ¼6, 120 mg/kg phlorizin-treated MCAO group: n¼ 7, 200 mg/kg phlorizin-treated MCAO group: n ¼12. (C) Results of the NDS on day 3 after MCAO. nnPo0.01, ##Po0.01. vehicle-treated sham group: n ¼12, 40 mg/kg phlorizin-treated sham group: n ¼6, 120 mg/kg phlorizin-treated sham group: n ¼4, 200 mg/kg phlorizin-treated sham group: n ¼6, vehicle-treated MCAO group: n¼ 17, 40 mg/kg phlorizin-treated MCAO group: n ¼ 6, 120 mg/kg phlorizin-treated MCAO group: n ¼ 7, 200 mg/kg phlorizin-treated MCAO group: n ¼12. veh: vehicle, PHZ: phlorizin, NDS: neurological deficit score.
2.5. Effect of phlorizin (i.c.v.) on the development of postischemic glucose intolerance in glucose (i.c.v.)-treated mice
2.7. Changes in the expression levels of cerebral SGLT-1 after cerebral ischemic stress
FBG were significantly increased on day 1 after MCAO in the saline-, mannitol- and glucose (10 or 25% (w/v))-treated group as compared with the sham group (Fig. 5). Phlorizin (40 mg/mouse) did not affect the elevation of FBG on day 1 after MCAO in the glucose (25% (w/v))-treated group (Fig. 5).
The changes in the expression levels of SGLT-1 were observed on day 1 after MCAO in the brain. The expression levels of SGLT-1 were significantly increased in the cortex and the striatum in the MCAO group than those of SGLT-1 in the sham group, but not changed in the hippocampus and the hypothalamus (Fig. 7).
2.6. Effect of phlorizin (i.c.v.) on the development of ischemic neuronal damage in glucose (i.c.v.)-treated mice
3.
The development of infarct volume was significantly and dose-dependently exacerbated on day 1 after MCAO in the glucose (10 or 25% (w/v))-treated group as compared with the saline-treated group. In contrast, mannitol alone did not affect infarct volume (Fig. 6A and B). Glucose (25% (w/v))induced exacerbation of infarct volume was significantly suppressed by phlorizin (40 mg/mouse) treatment on day 1 after MCAO as compared with the 25% (w/v) glucose-treated group (Fig. 6A and B). In addition, NDS was also significantly and dose-dependently exacerbated on day 1 after MCAO in the glucose (10 or 25% (w/v))-treated group (Fig. 6C). In addition, the glucose (25%(w/v))-induced increase of NDS was significantly decreased by phlorizin (40 mg/mouse) treatment on day 1 after MCAO (Fig. 6C).
As shown in our previous reports, ischemic neuronal damage can be triggered by glucose intolerance (post-ischemic hyperglycemia) that develops during the early phase of focal cerebral ischemic stress onset (Harada et al., 2009a, 2010). However, the developmental mechanism of neuronal damage by post-ischemic hyperglycemia is not clear. In our present study, by using phlorizin, an SGLT inhibitor, we confirmed that the SGLT family is involved in the development of ischemic neuronal damage. That is, neuronal damage following hyperglycemia was not observed following i.p. administration of phlorizin. It has been reported that the SGLT family exists in peripheral tissues, such as the small intestine and kidney (Glick and Mayer, 1968; Perez Lopez et al., 2010; Wright and Turk, 2004), and that both SGLT-1 and
Discussion
116
brain research 1489 (2012) 113–120
Fig. 3 – Effect of phlorizin (i.c.v.) on the elevation of FBG after cerebral ischemic stress. Mice were treated with phlorizin (10 or 40 lg/mouse, i.c.v.) immediately and 6 h after reperfusion. Effect of phlorizin on fasting blood glucose levels on day 1 after cerebral ischemia. Results are presented as the mean7S.E.M. nnPo0.01, nPo0.05, vehicle-treated sham group: n ¼ 8, 10 lg/mouse phlorizin-treated sham group: n¼ 6, 40 lg/mouse phlorizin-treated sham group: n ¼8, vehicle-treated MCAO group: n ¼16, 10 lg/mouse phlorizin-treated MCAO group: n ¼7, 40 lg/mouse phlorizin-treated MCAO group: n ¼18. veh: vehicle, PHZ: phlorizin.
Fig. 4 – Effect of phlorizin (i.c.v.) on the development of neuronal damage after cerebral ischemic stress. Representative photographs of TTC staining on day 3 after MCAO. (A) Quantitative analysis of the infarct volume. (B) Results are presented as the mean7S.E.M. ##Po0.01, vehicle-treated MCAO group: n¼ 16, 10 lg/mouse phlorizin-treated MCAO group: n ¼7, 40 lg/ mouse phlorizin-treated MCAO group: n ¼18. (C) Results of the NDS on day 3 after MCAO. nnPo0.01, ##Po0.01. vehicle-treated sham group: n ¼8, 10 lg/mouse phlorizin-treated sham group: n¼ 6, 40 lg/mouse phlorizin-treated sham group: n ¼ 8, vehicle-treated MCAO group: n ¼16, 10 lg/mouse phlorizin-treated MCAO group: n ¼7, 40 lg/mouse phlorizin-treated MCAO group: n¼ 18. veh: vehicle, PHZ: phlorizin, NDS: neurological deficit score. SGLT-2 expression increases in diabetic kidney (Malatiali et al., 2008). Interestingly, the presence of SGLT-1, 3, 4 and 6 have also been reported in various regions of the brain (O’Malley et al., 2006; Perez Lopez et al., 2010; Poppe et al.,
1997; Wright and Turk, 2004). However, their detailed function, except for peripheral areas, is not well understood. It is well known that neuronal cell death by stroke is involved in the induction of both apoptosis and necrosis, and that
brain research 1489 (2012) 113–120
117
Fig. 5 – Effect of phlorizin (i.c.v.) on the development of post-ischemic hyperglycemia in glucose (i.c.v.)-treated mice. Mice were treated with phlorizin (40 lg/mouse, i.c.v.) and/or glucose (10 or 25%, i.c.v.) immediately and 6 h after reperfusion. Effect of phlorizin and/or glucose on FBG on day 1 after cerebral ischemia. Results are presented as the mean7S.E.M. nnPo0.01, n Po0.05, saline-treated sham group: n¼ 8, 25% (w/v) glucose-treated sham group: n ¼ 4, saline-treated MCAO group: n¼ 10, mannitol-treated MCAO group: n ¼5, 10% (w/v) glucose-treated MCAO group: n ¼9, 25% (w/v) glucose-treated MCAO group: n ¼7, 25% (w/v) glucose and phlorizin-treated MCAO group: n ¼10. PHZ: phlorizin.
Fig. 6 – Effect of phlorizin (i.c.v.) on the development of ischemic neuronal damage in glucose (i.c.v.)-treated mice. Representative photographs of TTC staining on day 3 after MCAO. (A) Quantitative analysis of the infarct volume. (B) Results are presented as the mean7S.E.M. ##Po0.01, yyPo0.01, saline-treated MCAO group: n ¼ 10, mannitol-treated MCAO group: n ¼5, 10% (w/v) glucose-treated MCAO group: n ¼9, 25% (w/v) glucose-treated MCAO group: n ¼7, 25% (w/v) glucose and phlorizin-treated MCAO group: n ¼10. (C) Results of the NDS on day 3 after MCAO. nnPo0.01, #Po0.05, yPo0.05, sham group: n ¼8, saline-treated MCAO group: n ¼10, mannitol-treated MCAO group: n ¼ 5, 10% (w/v) glucose-treated MCAO group: n¼ 9, 25% glucose-treated MCAO group: n ¼7, 25% (w/v) glucose and phlorizin-treated MCAO group: n ¼10. PHZ: phlorizin, NDS: neurological deficit score. apoptosis occurs in the penumbral region (Dirnagl et al., 1999). Furthermore, neuronal damage following ischemic stroke in the penumbral region is reported to be affected by multiple factors, including glutamate-mediated excitotoxicity and inflammation leading to calcium overload, peri-infarct depolarization and oxidative stress (Dirnagl et al., 1999; Nakase et al., 2003; Shimmyo et al., 2008; Zhou et al., 2010). In contrast, high glucose conditions induce an early decrease in cell viability and an increase of apoptosis (Costa et al.,
2012; Moley et al., 1998). It has been shown that the SGLT family transports glucose together with Naþ ions into cells, and generates inward currents, resulting in depolarization and increased excitability (Burdakov et al., 2005). Therefore, the suppressive effect on neuronal damage by phlorizin may be mediated by inhibiting glucose-SGLT-induced depolarization in the brain. However, the systemic effect of phlorizin reduces blood glucose levels by inhibiting glucose absorption through SGLT-1 in the small intestine and glucose
118
brain research 1489 (2012) 113–120
Fig. 7 – Changes in the expression levels of cerebral SGLT-1 after cerebral ischemic stress. Representative western blots showing SGLT-1 and GAPDH levels in indicated brain regions (cortex, hippocampus, striatum and hypothalamus) on day 1 after MCAO. The relative levels were analyzed by determining the ratio of SGLT-1/GAPDH. Results are presented as the mean7S.E.M. nnPo0.01, vs. sham. sham group: n ¼ 9, MCAO group: n ¼11.
reabsorption though SGLT-2 in the kidney (Ehrenkranz et al., 2005; Kinne and Castaneda, 2011). We have also demonstrated that it is important to regulate systemic blood glucose levels after ischemic stress for the suppression of ischemic neuronal damage (Harada et al., 2009a). Therefore, the suppressive effect of post-ischemic hyperglycemia and neuronal damage by i.p. injection of phlorizin may be mainly affected by peripheral hypoglycemic effects. To confirm the central nervous system (CNS) effect of SGLTs, i.c.v. administration of phlorizin was performed. Ischemic neuronal damage was significantly suppressed by i.c.v. administration of phlorizin, as well as i.p. administration of phlorizin. Interestingly, i.c.v. administration of phlorizin did not affect the ischemic stress-induced increase of FBG. Therefore, the CNS SGLT family may be directly involved in the development of ischemic neuronal damage. We further examined the relationship between glucose and the SGLT family by co-administering glucose and phlorizin. In the present study, high glucose conditions achieved by i.c.v. injection of 25% (w/v) glucose solution in the brain dramatically exacerbated the development of ischemic neuronal damage without affecting the ischemic stress-induced increase of FBG. In general, hyperosmolar pressure agents are used in the treatment of cerebral stroke. However, administration of 27.5% (w/v) mannitol solution, which has the same osmotic pressure as 25% (w/v) glucose solution, had no effect on the development of ischemic neuronal damage, suggesting that osmotic pressure has no bearing on the exacerbative effect of 25% (w/v) glucose solution. These results indicate that post-ischemic elevations of glucose levels enhance ischemic neuronal damage and that exacerbation of ischemic neuronal damage is mediated by the cerebral SGLT family. Hence, we hypothesize that the development of post-ischemic hyperglycemia may induce excessive activation of the SGLT family leading to depolarization. It has been shown that glutamate receptors, such as N-methyl-D-aspartate receptors and a-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid receptors, and voltagedependent calcium channels, play an important role in the influx of Ca2þ into the cell (McDonald et al., 1998; Morikawa et al., 1998). As a consequence, ischemic neuronal damage may be induced by enhancing glutamic acid release from excitatory neurons and/or Ca2þ accumulation in cells by
voltage-dependent calcium channel opening through glucose-induced stimulation of the SGLT family. Moreover, the influx of intracellular glucose mediates ischemic neuronal damage through the SGLT family, which may be enhanced by adenosine tri-phosphate and reactive oxygen species produced by the metabolism of glucose. In addition, these phenomena may be involved in the ischemic stressinduced increase of SGLT-1 in cerebral ischemic stresssensitive organs such as the cortex and the striatum. In conclusion, cerebral SGLTs seem to be activated by postischemic hyperglycemia and may be involved in the exacerbation of ischemic neuronal damage. Our results from the present study may be helpful for the development of new agents and strategies for clinical treatment of cerebral stroke.
4.
Experimental procedures
4.1.
Animals
All experiments were performed on male ddY mice (5 weeks old, 25–30 g) obtained from SLC (Shizuoka, Japan). The animals were housed at 23–24 1C with a 12-h light–dark cycle (lights on 8:00 a.m. to 8:00 p.m.). Food and water were available ad libitum. The present study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, adopted by the Japanese Pharmacological Society. In addition, all experiments were approved by the ethical committee for animals of Kobe Gakuin University (approval number: A060601-10).
4.2.
Animal models of focal cerebral ischemia
The experimental transient focal ischemia mouse model was generated by performing MCAO as described previously (Harada et al., 2009b). Briefly, mice were anesthetized with isoflurane and kept under a heating lamp to maintain core body temperature at 37.070.5 1C. The left middle cerebral artery was occluded for 2 h by the insertion of 8–0 nylon monofilament with a thin silicon coat through the common carotid artery followed by reperfusion. Sham-operated mice underwent the same surgical procedure without suture insertion. Relative cerebral blood flow was measured by laser
brain research 1489 (2012) 113–120
119
Doppler flowmetry (LDF; TBF-LN1, Unique Medical, Osaka, Japan) to assess the adequacy of vascular occlusion and reperfusion, as described previously (Harada et al., 2009b). Physiological parameters were measured before, during and 30 min after MCAO by using a sphygmomanometer (TK-370C; Brain Science idea, Osaka, Japan) and an i-STAT (300F; FUSO Pharmaceutical Industries, Osaka, Japan), as described previously (Harada et al., 2009b). The relative cerebral blood flow and physiological parameters were not altered in this study.
Japan) or vehicle immediately after reperfusion. In the second experiment, mice were i.c.v. administered with 10 or 40 mg/ mouse phlorizin or vehicle immediately and 6 h after reperfusion. In the third experiment, mice were i.c.v. administered with 10 or 25% (w/v) glucose (Nakalai Tesque, Kyoto, Japan), 40 mg/mouse phlorizin, 27.5% (w/v) mannitol (Wako, Osaka, Japan) and saline immediately and 6 h after reperfusion. All i.c.v. administrations were performed as previously described (Harada et al., 2011).
4.3.
4.7.
Measurement of infarct volume
Mice were decapitated and brains were dissected on day 1 after MCAO (i.c.v. administration of glucose and/or phlorizin) or day 3 after MCAO (i.p. administration of phlorizin). The infarct volume of the entire brain represented the degree of cerebral infarction. The brains were cut into 2 mm thick coronal slices. The brain slices were incubated in saline containing 2% (w/v) 2, 3, 5-triphenyltetrazolium chloride (TTC; Sigma, MO, USA) for 10 min at 37 1C. The stained slices were fixed with 4% (w/v) paraformaldehyde (Sigma), and the infarct volumes were measured using image analysis software (ImageJ (NIH, MD, USA) and adobe photoshop elements 5.0 (Adobe Systems Incorporated, Tokyo, Japan)) as previously described (Harada et al., 2009a, 2009b). The infarct volume (mm3) was calculated by multiplication of infarct volume (mm3) and intensity (intensity¼ intensity of left hemisphereintensity of right hemisphere).
4.4.
Neurological examination
Neurological examination was performed after reperfusion using the following NDS: consciousness (0, normal; 1, restless; 2, lethargic; 3, stuporous; 4, seizures; and 5, death), walking (0, normal; 1, paw; 2, unbalanced walking; 3, circling; 4, unable to stand; and 5, no movement), limb tone (0, normal; 1, spastic; and 2, flaccid) and pain reflex (Harada et al., 2009a, 2009b). Pain reflex was assessed using the tail flick test (pain reflex¼ latency after MCAO latency before MCAO). Pain reflex means NDS points. A cut-off time of 10 s was used to prevent any injury to the tail. NDS is added pain reflex in total score of consciousness, walking, and limb tone. In this study, the NDS was examined on day 1 after MCAO (i.c.v. administration of glucose and/or phlorizin) or on day 3 after MCAO (i.p. administration of phlorizin).
4.5.
Measurement of FBG
Mice were fasted for 15 h until measurement of FBG, and approximately 1.5-mL of blood was obtained from the tail vein. Plasma FBG was measured using the glucose pilot (Aventir Biotech, CA, USA). The increment of FBG was calculated using the following formula: increment of FBG¼FBG after MCAO-FBG before MCAO. In this study, FBG was measured on day 1 after MCAO.
Western blot analysis
Western blotting was performed as previously described but with some modifications (Harada et al., 2009b, 2010, 2011). Briefly, the cortex, hippocampus, striatum and hypothalamus were homogenized in homogenization buffer and protein samples (20 mg) were electrophoresed on 7.5% (w/v) sodium dodecyl sulfate polyacrylamide gels. Protein was transferred onto nitrocellulose membranes (BioRad, CA, USA). SGLT-1 was detected using the corresponding primary antibodies from Millipore (MA, USA, 1:1000). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control and was detected using primary antibodies from Millipore (1:20,000). Blots for SGLT-1 and GAPDH were incubated overnight with the primary antibody at 4 1C in Tris-buffered saline containing 1% (v/v) Tween-20 and 5% (w/v) blocking agent (GE Healthcare, Tokyo, Japan). After washing, blots were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:1000; KPL, Guildford, UK) for SGLT-1, or HRP-conjugated anti-mouse IgG (1:10000; KPL) for GAPDH for 1 h at room temperature. All visualization of immunoreactive bands was performed using Light-Capture (AE-6981; ATTO, Tokyo, Japan) with an ECLTM Western blotting analysis system (GE Healthcare). The signal intensity of immunoreactive bands was analyzed using a CsAnalyzer (Ver. 3.0; ATTO).
4.8.
Statistical analysis
The infarct volume and FBG levels were analyzed using oneway analysis of variance followed by Scheffe’s test. Results of western blots were analyzed using unpaired Student’s t-tests. The data are presented as the mean7standard error of the mean (S.E.M.). NDS was analyzed using the Steel–Dwass test with post-hoc nonparametric multiple comparison tests, and the data are presented as the medians (25th–75th percentile). A P value of o0.05 was regarded as significant.
Acknowledgments This study was supported by Grants-in-Aid and by special coordination funds from Grants-in-Aid for Scientific Research (C) (22500683) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
r e f e r e nc e s 4.6.
Administration of phlorizin and glucose
In the first experiment, mice were i.p. administered with 40, 120, or 200 mg/kg phlorizin (Tokyo Chemical Industry, Tokyo,
Benchoua, A., Guegan, C., Couriaud, C., Hosseini, H., Sampaio, N., Morin, D., Onteniente, B., 2001. Specific caspase pathways are
120
brain research 1489 (2012) 113–120
activated in the two stages of cerebral infarction. J. Neurosci. 21, 7127–7134. Burdakov, D., Luckman, S.M., Verkhratsky, A., 2005. Glucosesensing neurons of the hypothalamus. Philos. Trans. R. Soc. Lond. B: Biol. Sci. 360, 2227–2235. Costa, G.N., Vindeirinho, J., Cavadas, C., Ambrosio, A.F., Santos, P.F., 2012. Contribution of TNF receptor 1 to retinal neural cell death induced by elevated glucose. Mol. Cell. Neurosci. 50, 113–123. Diez-Sampedro, A., Hirayama, B.A., Osswald, C., Gorboulev, V., Baumgarten, K., Volk, C., Wright, E.M., Koepsell, H., 2003. A glucose sensor hiding in a family of transporters. Proc. Nat. Acad. Sci. U.S.A. 100, 11753–11758. Dirnagl, U., Iadecola, C., Moskowitz, M.A., 1999. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 22, 391–397. Ehrenkranz, J.R., Lewis, N.G., Kahn, C.R., Roth, J., 2005. Phlorizin: a review. Diabetes Metab. Res. Rev. 21, 31–38. Fuentes, B., Castillo, J., San Jose, B., Leira, R., Serena, J., Vivancos, J., Davalos, A., Nunez, A.G., Egido, J., Diez-Tejedor, E., 2009. The prognostic value of capillary glucose levels in acute stroke: the GLycemia in acute stroke (GLIAS) study. Stroke 40, 562–568. Glick, Z., Mayer, J., 1968. Hyperphagia caused by cerebral ventricular infusion of phloridzin. Nature 219, 1374. Harada, S., Fujita, W.H., Shichi, K., Tokuyama, S., 2009a. The development of glucose intolerance after focal cerebral ischemia participates in subsequent neuronal damage. Brain Res. 1279, 174–181. Harada, S., Hamabe, W., Kamiya, K., Satake, T., Yamamoto, J., Tokuyama, S., 2009b. Preventive effect of Morinda citrifolia fruit juice on neuronal damage induced by focal ischemia. Biol. Pharm. Bull. 32, 405–409. Harada, S., Fujita-Hamabe, W., Tokuyama, S., 2010. The importance of regulation of blood glucose levels through activation of peripheral 50 -AMP-activated protein kinase on ischemic neuronal damage. Brain Res. 1351, 254–263. Harada, S., Fujita-Hamabe, W., Tokuyama, S., 2011. Effect of orexin-A on post-ischemic glucose intolerance and neuronal damage. J. Pharmacol. Sci. 115, 155–163. Harada, S., Fujita-Hamabe, W., Tokuyama, S., 2012. Ischemic stroke and glucose intolerance: a review of the evidence and exploration of novel therapeutic targets. J. Pharmacol. Sci. 118, 1–13. Kernan, W.N., Viscoli, C.M., Inzucchi, S.E., Brass, L.M., Bravata, D.M., Shulman, G.I., McVeety, J.C., 2005. Prevalence of abnormal glucose tolerance following a transient ischemic attack or ischemic stroke. Arch. Intern. Med. 165, 227–233. Kinne, R.K., Castaneda, F., 2011. SGLT inhibitors as new therapeutic tools in the treatment of diabetes. Handb. Exp. Pharmacol., 105–126. Malatiali, S., Francis, I., Barac-Nieto, M., 2008. Phlorizin prevents glomerular hyperfiltration but not hypertrophy in diabetic rats. Exp. Diabetes Res. 2008, 305403. Matz, K., Keresztes, K., Tatschl, C., Nowotny, M., Dachenhausenm, A., Brainin, M., Tuomilehto, J., 2006. Disorders of glucose metabolism
in acute stroke patients: an underrecognized problem. Diabetes Care 29, 792–797. McDonald, J.W., Althomsons, S.P., Hyrc, K.L., Choi, D.W., Goldberg, M.P., 1998. Oligodendrocytes from forebrain are highly vulnerable to AMPA/kainate receptor-mediated excitotoxicity. Nat. Med. 4, 291–297. Moley, K.H., Chi, M.M., Knudson, C.M., Korsmeyer, S.J., Mueckler, M.M., 1998. Hyperglycemia induces apoptosis in pre-implantation embryos through cell death effector pathways. Nat. Med. 4, 1421–1424. Morikawa, E., Mori, H., Kiyama, Y., Mishina, M., Asano, T., Kirino, T., 1998. Attenuation of focal ischemic brain injury in mice deficient in the epsilon1 (NR2A) subunit of NMDA receptor. J. Neurosci. 18, 9727–9732. Nakase, T., Fushiki, S., Naus, C.C., 2003. Astrocytic gap junctions composed of connexin 43 reduce apoptotic neuronal damage in cerebral ischemia. Stroke 34, 1987–1993. O’Malley, D., Reimann, F., Simpson, A.K., Gribble, F.M., 2006. Sodium-coupled glucose cotransporters contribute to hypothalamic glucose sensing. Diabetes 55, 3381–3386. Pellegrini-Giampietro, D.E., Gorter, J.A., Bennett, M.V., Zukin, R.S., 1997. The GluR2 (GluR-B) hypothesis: Ca(2þ)-permeable AMPA receptors in neurological disorders. Trends Neurosci. 20, 464–470. Perez Lopez, G., Gonzalez Albarran, O., Cano Megias, M., 2010. Sodium-glucose cotransporter type 2 inhibitors (SGLT2): from familial renal glucosuria to the treatment of type 2 diabetes mellitus. Nefrologia 30, 618–625. Poppe, R., Karbach, U., Gambaryan, S., Wiesinger, H., Lutzenburg, M., Kraemer, M., Witte, O.W., Koepsell, H., 1997. Expression of the Naþ-D-glucose cotransporter SGLT1 in neurons. J. Neurochem. 69, 84–94. Saito, I., 2012. Epidemiological evidence of type 2 diabetes mellitus, metabolic syndrome, and cardiovascular disease in Japan. Circ. J. 76, 1066–1073. Scheepers, A., Joost, H.G., 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. Shimmyo, Y., Kihara, T., Akaike, A., Niidome, T., Sugimoto, H., 2008. Three distinct neuroprotective functions of myricetin against glutamate-induced neuronal cell death: involvement of direct inhibition of caspase-3. J. Neurosci. Res. 86, 1836–1845. Shuaib, A., Lees, K.R., Lyden, P., Grotta, J., Davalos, A., Davis, S.M., Diener, H.C., Ashwood, T., Wasiewski, W.W., Emeribe, U., 2007. NXY-059 for the treatment of acute ischemic stroke. N. Engl. J. Med. 357, 562–571. Wright, E.M., Turk, E., 2004. The sodium/glucose cotransport family SLC5. Pflugers Arch. 447, 510–518. Zhou, L., Li, F., Xu, H.B., Luo, C.X., Wu, H.Y., Zhu, M.M., Lu, W., Ji, X., Zhou, Q.G., Zhu, D.Y., 2010. Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95. Nat. Med. 16, 1439–1443.
