Metab Brain Dis (2014) 29:47–58 DOI 10.1007/s11011-013-9475-2
ORIGINAL PAPER
Activation of AMP-activated protein kinase by metformin protects against global cerebral ischemia in male rats: Interference of AMPK/PGC-1α pathway Ghorbangol Ashabi & Fariba Khodagholi & Leila Khalaj & Mahdi Goudarzvand & Masoumeh Nasiri
Received: 12 October 2013 / Accepted: 16 December 2013 / Published online: 18 January 2014 # Springer Science+Business Media New York 2014
Abstract Here, we have investigated the effect of metformin pretreatment in the rat models of global cerebral ischemia. Cerebral ischemia which leads to brain dysfunction is one of the main causes of neurodegeneration and death worldwide. Metformin is used in clinical drug therapy protocols of diabetes. It is suggested that metformin protects cells under hypoxia and ischemia in non-neuronal contexts. Protective effects of metformin may be modulated via activating the AMP activated protein kinase (AMPK). Our results showed that induction of 30 min global cerebral I/R injury using 4-vesseles occlusion model led to significant cell death in the rat brain. Metformin pretreatment (200 mg kg/once/day, p.o., 2 weeks) attenuated apoptotic cell death and induced mitochondrial biogenesis proteins in the ischemic rats, analyzed using histological and Western blot assays. Besides, inhibition of AMPK by compound c showed that metformin resulted in apoptosis attenuation via AMPK activation. Interestingly, AMPK activation was also involved in the induction of mitochondrial
G. Ashabi Department of Physiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran F. Khodagholi : M. Nasiri NeuroBiology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran F. Khodagholi : M. Nasiri Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran L. Khalaj : M. Goudarzvand Medical School, Alborz University of Medical Sciences, Alborz, Iran L. Khalaj (*) Physiology-Pharmacology Department, Medical School, Alborz University of Medical Sciences, P.O. Box: 3148/561 Eshteraki Ave., Baghestan Blvd., Karaj, Iran e-mail: [email protected]
biogenesis proteins using metformin, inhibition of AMPK by compound c reversed such effect, further supporting the role of AMPK upstream of mitochondrial biogenesis proteins. In summary, Metformin pretreatment is able to modulate mitochondrial biogenesis and apoptotic cell death pathways through AMPK activation in the context of global cerebral ischemia, conducting the outcome towards neuroprotection. Keyword Global cerebral ischemia . Metformin . AMPK . Mitochondrial biogenesis proteins . Apoptosis . Compound c
Introduction Cerebral ischemia which leads to brain dysfunction is one of the main causes of neurodegeneration and death worldwide (Li et al. 2007). Ischemia/reperfiusion injury (I/R) enhances reactive oxygen species (ROS) level, as well as apoptosis in neuronal cells (Wang et al. 2007). Scientists have used experimental animal models of stroke and global cerebral ischemia investigating the protective efficiency of various drugs to be used in humans. Metformin is introduced in clinical drug therapy protocols of diabetes for decades. It has been well established that metformin decreases blood glucose level by enhancing insulin receptor sensitivity (Kirpichnikov et al. 2002). Protective effect of metformin against cardiac I/R and heart failure is well recognized (Yeh et al. 2011). It is suggested that protective effects of metformin may be modulated via activating the AMP activated protein kinase (AMPK). In several stressful situations, when cellular ATP levels fall down, the AMPK is phosphorylated and consequently activates its related proteins (Mukherjee et al. 2008). In ischemic condition, the Thr172 in the catalytic subunit of AMPK protein is reported to be phosphorylated. Then phosphorylated AMPK increases glucose uptake and cellular energy for metabolism (Atherton et al. 2005; Horman et al. 2006). There are a wide range of
48
scientific papers reporting that metformin could decrease apoptotic cell death in vitro and in vivo models of hypoxic and ischemic condition in skeletal muscle, liver and heart (Vytla and Ochs 2013; Viollet and Foretz 2013; Kristensen et al. 2013). Although reports regarding the protective role of metformin in different neuronal contexts and the involved mechanisms are less available (Pintana et al. 2012; Ullah et al. 2012). Protective effect of metformin is to somehow attributed to inhibition of mitochondrial mediated apoptosis pathway. El-mir and colleagues have demonstrated that metformin can inhibit cytochrome c release from mitochondrial membrane (El-Mir et al. 2008). In cardiac cells, activation of AMPK by metformin promotes mitochondrial biogenesis via some downstream proteins such as proliferator-activated receptor gamma coactivator-1α (PGC-1α) and mitochondrial transcription factor A (TFAM) (Irrcher et al. 2003). Activation of mitochondrial biogenesis proteins are shown to be involved in neuronal cell protection via enhancing antioxidant enzyme activities (Mohagheghi et al. 2012). Induction of mitochondrial biogenesis proteins has also shown protection against rat global cerebral I/R injury (Chen et al. 2011; Zhang et al. 2012; Mohagheghi et al. 2013). In order to unravel more of metformin potential in the neuronal context, here we investigated metformin pretreatment effects in the rat models of global cerebral ischemia, mainly focusing on AMPK/mitochondrial biogenesis, as well as apoptosis proteins.
Experimental procedure Animals Six month male Wister rats were housed in standard cages under controlled temperature (22±2 °C), humidity and a 12 h light/dark cycle (light on 07:00–19:00), with food and water provided ad libitum. Experimentation was approved by the Ethics Committee of Shahid Beheshti Medical University in accordance with international guidelines for animal experiments. All efforts were made to minimize animal suffering, and to reduce the number of animals used. Mortality rates didn’t show significant changes between experimental groups which underwent ischemia (6 of 12). During experiment one of the animals which received 400 mg/kg metformin was dead before ischemia induction, and no statistically significant changes in the animals’ weights and blood glucose levels were detected.
Metab Brain Dis (2014) 29:47–58
200 and 400 mg/kg once daily for 2 weeks, then underwent cerebral ischemia using 4-vessles occlusion model. The last gavage was performed 24 h before induction of ischemia. In the last step, animals were divided into 5 group; sham, ischemia, 200 mg/kg metformin, “200 mg/kg metformin + ischemia (30 min)”, “Compound c; as an AMPK inhibitor (Sigma) + 200 mg/kg metformin + ischemia (30 min)” (Fig. 1). Preparation of compound c Compound c was dissolved in DMSO 10 %, and aliquots were stored at −20 °C until use. Aliquots of compound c at a concentration of 5 μg/μl prepared in Phosphate Buffer Saline (PBS, 0.1 M) for single intravenous (5 μl) injection at 30 min before ischemia. Surgery procedure Rats underwent transient forebrain global ischemia as described by Pulsinelli et al. (Pulsinelli and Brierley 1979). Briefly, on the first day, rats were anesthetized by chloral hydrate (400 mg/kg). A sterile string was loosely placed around each common carotid artery (CCA) without interrupting carotid blood flow and the incision was sutured. Both vertebral arteries were permanently electrocoagulated. Electroencephalogram (EEG) electrodes were fixed bilaterally at the skull on the parietal cortex. On the second day, under chloral hydrate anesthesia both CCA were occluded for 30 min. Rats with 4VO were only included if EEG was flattening during ischemia (Diler et al. 2002). Seventy-two hours reperfusion was initiated by opening the carotid clamps after 30 min of ischemia. Sham surgery involved exposure of common carotid and vertebral arteries. Rectal temperature was monitored (Citizen-513w) and kept at 37 °C by surface heating and cooling during surgery. Sacrifice and tissue preparation After 72 h reperfusion, each group (n=10) was split into two subgroups. The animals within the first subgroup (n=4) were perfused transcardially with phosphate-buffered saline (PBS) (pH 7.4), followed by 4 % paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) under chloral hydrate anesthesia (400 mg/kg i.p.). The whole brains were then removed and
Experimental design At the first step of experimental design, animals were divided into four groups; Sham, 15, 20 and 30 min ischemia. In the second step, animals were pre-treated with metformin (Osvah Pharmaceutical Co, Tehran, Iran) in 3 different doses: 100,
Fig. 1 Flow chart showing the time line scheme of the experiment
Metab Brain Dis (2014) 29:47–58
post fixed in 4 % paraformaldehyde for 24 h and subsequently embedded in paraffin for histopathologic studies. Both left and right hippocampi were evaluated by hematoxilin and eosin staining. However, in figures just the sections obtained from right side are represented. In the second subgroup animals (n=6) were sacrificed by CO2 asphyxiation. Rats were decapitated, brains removed, and both hippocampi isolated on ice, frozen in liquid nitrogen and stored at −80 °C for western blot analysis. Hematoxiline and Eosin (H&E) staining Whole brain tissue embedded in paraffin was used for further histopathologic preparations. Coronal sections (4–5 lm thickness) of hippocampal formation were prepared and stained with H&E. Degenerated cells can be inferred based on the morphological changes: neuronal shrinkage, vacuolation, and hyperchromatism. Neurons were counted when a clearly visible nucleolus located within the counting frame came into focus. Cells without nucleoli or with nucleoli that intersected the lower or left edge of the counting frame were excluded. To obtain the mean percentage of damaged neurons within the CA1 subfield of the dorsal hippocampus, the damaged neurons from the total number of neurons was counted on three adjacent 400× microscopic images.
49
follows; Rabbit and mouse IgG-HRP-linked antibodies (Cell Signaling; 1/1,000). Blots were revealed by ECL advanced kit (Amersham Biosciences). To normalize for protein content, blots were stripped in stripping buffer containing 100 mM 2mercaptoethanol, 2 % (w/v) SDS, 62.5 mM Tris–HCl (pH= 6.7) and then probed with anti β-actin antibody (Cell Signaling; 1/1,000). The density of bands was quantified using NIH Image J, and the ratio to β-actin was calculated. Western blotting was used to measure the protein expression of PGC1α (ABCAM; 1 μg/ml), NRF-1(Santa Cruz; 1/1,000) and TFAM (BioVision; 0.5 μg/ml) as major proteins involved in mitochondrial biogenesis. The expressions of cleaved caspase-3, cleaved PARP, Bax and Bcl-2 (Cell Signaling Technology; 1/1,000) as markers of apoptosis as well as AMPK proteins (Cell Signaling Technology; 1/1,000) were measured. Statistical analysis The number of neurons within the hippocampus was analyzed using a non-parametric method, Mann–Whitney U test. Western blot data was analyzed by a oneway analysis of variance (ANOVA) followed by Tukey HSD for multiple comparisons, using SPSS 16.0 package programs. Data are expressed as mean ± SEM and statistical significance was set at P
ORIGINAL PAPER
Activation of AMP-activated protein kinase by metformin protects against global cerebral ischemia in male rats: Interference of AMPK/PGC-1α pathway Ghorbangol Ashabi & Fariba Khodagholi & Leila Khalaj & Mahdi Goudarzvand & Masoumeh Nasiri
Received: 12 October 2013 / Accepted: 16 December 2013 / Published online: 18 January 2014 # Springer Science+Business Media New York 2014
Abstract Here, we have investigated the effect of metformin pretreatment in the rat models of global cerebral ischemia. Cerebral ischemia which leads to brain dysfunction is one of the main causes of neurodegeneration and death worldwide. Metformin is used in clinical drug therapy protocols of diabetes. It is suggested that metformin protects cells under hypoxia and ischemia in non-neuronal contexts. Protective effects of metformin may be modulated via activating the AMP activated protein kinase (AMPK). Our results showed that induction of 30 min global cerebral I/R injury using 4-vesseles occlusion model led to significant cell death in the rat brain. Metformin pretreatment (200 mg kg/once/day, p.o., 2 weeks) attenuated apoptotic cell death and induced mitochondrial biogenesis proteins in the ischemic rats, analyzed using histological and Western blot assays. Besides, inhibition of AMPK by compound c showed that metformin resulted in apoptosis attenuation via AMPK activation. Interestingly, AMPK activation was also involved in the induction of mitochondrial
G. Ashabi Department of Physiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran F. Khodagholi : M. Nasiri NeuroBiology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran F. Khodagholi : M. Nasiri Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran L. Khalaj : M. Goudarzvand Medical School, Alborz University of Medical Sciences, Alborz, Iran L. Khalaj (*) Physiology-Pharmacology Department, Medical School, Alborz University of Medical Sciences, P.O. Box: 3148/561 Eshteraki Ave., Baghestan Blvd., Karaj, Iran e-mail: [email protected]
biogenesis proteins using metformin, inhibition of AMPK by compound c reversed such effect, further supporting the role of AMPK upstream of mitochondrial biogenesis proteins. In summary, Metformin pretreatment is able to modulate mitochondrial biogenesis and apoptotic cell death pathways through AMPK activation in the context of global cerebral ischemia, conducting the outcome towards neuroprotection. Keyword Global cerebral ischemia . Metformin . AMPK . Mitochondrial biogenesis proteins . Apoptosis . Compound c
Introduction Cerebral ischemia which leads to brain dysfunction is one of the main causes of neurodegeneration and death worldwide (Li et al. 2007). Ischemia/reperfiusion injury (I/R) enhances reactive oxygen species (ROS) level, as well as apoptosis in neuronal cells (Wang et al. 2007). Scientists have used experimental animal models of stroke and global cerebral ischemia investigating the protective efficiency of various drugs to be used in humans. Metformin is introduced in clinical drug therapy protocols of diabetes for decades. It has been well established that metformin decreases blood glucose level by enhancing insulin receptor sensitivity (Kirpichnikov et al. 2002). Protective effect of metformin against cardiac I/R and heart failure is well recognized (Yeh et al. 2011). It is suggested that protective effects of metformin may be modulated via activating the AMP activated protein kinase (AMPK). In several stressful situations, when cellular ATP levels fall down, the AMPK is phosphorylated and consequently activates its related proteins (Mukherjee et al. 2008). In ischemic condition, the Thr172 in the catalytic subunit of AMPK protein is reported to be phosphorylated. Then phosphorylated AMPK increases glucose uptake and cellular energy for metabolism (Atherton et al. 2005; Horman et al. 2006). There are a wide range of
48
scientific papers reporting that metformin could decrease apoptotic cell death in vitro and in vivo models of hypoxic and ischemic condition in skeletal muscle, liver and heart (Vytla and Ochs 2013; Viollet and Foretz 2013; Kristensen et al. 2013). Although reports regarding the protective role of metformin in different neuronal contexts and the involved mechanisms are less available (Pintana et al. 2012; Ullah et al. 2012). Protective effect of metformin is to somehow attributed to inhibition of mitochondrial mediated apoptosis pathway. El-mir and colleagues have demonstrated that metformin can inhibit cytochrome c release from mitochondrial membrane (El-Mir et al. 2008). In cardiac cells, activation of AMPK by metformin promotes mitochondrial biogenesis via some downstream proteins such as proliferator-activated receptor gamma coactivator-1α (PGC-1α) and mitochondrial transcription factor A (TFAM) (Irrcher et al. 2003). Activation of mitochondrial biogenesis proteins are shown to be involved in neuronal cell protection via enhancing antioxidant enzyme activities (Mohagheghi et al. 2012). Induction of mitochondrial biogenesis proteins has also shown protection against rat global cerebral I/R injury (Chen et al. 2011; Zhang et al. 2012; Mohagheghi et al. 2013). In order to unravel more of metformin potential in the neuronal context, here we investigated metformin pretreatment effects in the rat models of global cerebral ischemia, mainly focusing on AMPK/mitochondrial biogenesis, as well as apoptosis proteins.
Experimental procedure Animals Six month male Wister rats were housed in standard cages under controlled temperature (22±2 °C), humidity and a 12 h light/dark cycle (light on 07:00–19:00), with food and water provided ad libitum. Experimentation was approved by the Ethics Committee of Shahid Beheshti Medical University in accordance with international guidelines for animal experiments. All efforts were made to minimize animal suffering, and to reduce the number of animals used. Mortality rates didn’t show significant changes between experimental groups which underwent ischemia (6 of 12). During experiment one of the animals which received 400 mg/kg metformin was dead before ischemia induction, and no statistically significant changes in the animals’ weights and blood glucose levels were detected.
Metab Brain Dis (2014) 29:47–58
200 and 400 mg/kg once daily for 2 weeks, then underwent cerebral ischemia using 4-vessles occlusion model. The last gavage was performed 24 h before induction of ischemia. In the last step, animals were divided into 5 group; sham, ischemia, 200 mg/kg metformin, “200 mg/kg metformin + ischemia (30 min)”, “Compound c; as an AMPK inhibitor (Sigma) + 200 mg/kg metformin + ischemia (30 min)” (Fig. 1). Preparation of compound c Compound c was dissolved in DMSO 10 %, and aliquots were stored at −20 °C until use. Aliquots of compound c at a concentration of 5 μg/μl prepared in Phosphate Buffer Saline (PBS, 0.1 M) for single intravenous (5 μl) injection at 30 min before ischemia. Surgery procedure Rats underwent transient forebrain global ischemia as described by Pulsinelli et al. (Pulsinelli and Brierley 1979). Briefly, on the first day, rats were anesthetized by chloral hydrate (400 mg/kg). A sterile string was loosely placed around each common carotid artery (CCA) without interrupting carotid blood flow and the incision was sutured. Both vertebral arteries were permanently electrocoagulated. Electroencephalogram (EEG) electrodes were fixed bilaterally at the skull on the parietal cortex. On the second day, under chloral hydrate anesthesia both CCA were occluded for 30 min. Rats with 4VO were only included if EEG was flattening during ischemia (Diler et al. 2002). Seventy-two hours reperfusion was initiated by opening the carotid clamps after 30 min of ischemia. Sham surgery involved exposure of common carotid and vertebral arteries. Rectal temperature was monitored (Citizen-513w) and kept at 37 °C by surface heating and cooling during surgery. Sacrifice and tissue preparation After 72 h reperfusion, each group (n=10) was split into two subgroups. The animals within the first subgroup (n=4) were perfused transcardially with phosphate-buffered saline (PBS) (pH 7.4), followed by 4 % paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) under chloral hydrate anesthesia (400 mg/kg i.p.). The whole brains were then removed and
Experimental design At the first step of experimental design, animals were divided into four groups; Sham, 15, 20 and 30 min ischemia. In the second step, animals were pre-treated with metformin (Osvah Pharmaceutical Co, Tehran, Iran) in 3 different doses: 100,
Fig. 1 Flow chart showing the time line scheme of the experiment
Metab Brain Dis (2014) 29:47–58
post fixed in 4 % paraformaldehyde for 24 h and subsequently embedded in paraffin for histopathologic studies. Both left and right hippocampi were evaluated by hematoxilin and eosin staining. However, in figures just the sections obtained from right side are represented. In the second subgroup animals (n=6) were sacrificed by CO2 asphyxiation. Rats were decapitated, brains removed, and both hippocampi isolated on ice, frozen in liquid nitrogen and stored at −80 °C for western blot analysis. Hematoxiline and Eosin (H&E) staining Whole brain tissue embedded in paraffin was used for further histopathologic preparations. Coronal sections (4–5 lm thickness) of hippocampal formation were prepared and stained with H&E. Degenerated cells can be inferred based on the morphological changes: neuronal shrinkage, vacuolation, and hyperchromatism. Neurons were counted when a clearly visible nucleolus located within the counting frame came into focus. Cells without nucleoli or with nucleoli that intersected the lower or left edge of the counting frame were excluded. To obtain the mean percentage of damaged neurons within the CA1 subfield of the dorsal hippocampus, the damaged neurons from the total number of neurons was counted on three adjacent 400× microscopic images.
49
follows; Rabbit and mouse IgG-HRP-linked antibodies (Cell Signaling; 1/1,000). Blots were revealed by ECL advanced kit (Amersham Biosciences). To normalize for protein content, blots were stripped in stripping buffer containing 100 mM 2mercaptoethanol, 2 % (w/v) SDS, 62.5 mM Tris–HCl (pH= 6.7) and then probed with anti β-actin antibody (Cell Signaling; 1/1,000). The density of bands was quantified using NIH Image J, and the ratio to β-actin was calculated. Western blotting was used to measure the protein expression of PGC1α (ABCAM; 1 μg/ml), NRF-1(Santa Cruz; 1/1,000) and TFAM (BioVision; 0.5 μg/ml) as major proteins involved in mitochondrial biogenesis. The expressions of cleaved caspase-3, cleaved PARP, Bax and Bcl-2 (Cell Signaling Technology; 1/1,000) as markers of apoptosis as well as AMPK proteins (Cell Signaling Technology; 1/1,000) were measured. Statistical analysis The number of neurons within the hippocampus was analyzed using a non-parametric method, Mann–Whitney U test. Western blot data was analyzed by a oneway analysis of variance (ANOVA) followed by Tukey HSD for multiple comparisons, using SPSS 16.0 package programs. Data are expressed as mean ± SEM and statistical significance was set at P
