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Metabolic syndrome has deleterious effects on the central nervous system, and recent evidence suggests that obesity rates are higher at presentation in children who develop epilepsy. Adiponectin is secreted by adipose tissue and acts in the brain and peripheral organs to regulate glucose and lipid metabolism. Adiponectin deficiency predisposes toward metabolic syndrome, characterized by obesity, insulin resistance, impaired glucose tolerance, hyperlipidemia, and cardiovascular morbidity. To investigate the relationship between metabolic syndrome and seizures, wild type C57BL/6J and adiponectin knockout mice were fed a high-fat diet, followed by treatment with low doses of kainic acid to induce seizures. Adiponectin deficiency in mice fed a high-fat diet resulted in greater fat accumulation, impaired glucose tolerance, hyperlipidemia, increased seizure severity and increased hippocampal pathology. In contrast, there were no adverse effects of adiponectin deficiency on metabolic phenotype or seizure activity in mice fed a normal (low fat) chow diet. These findings demonstrate that metabolic syndrome modulates the outcome of seizures and brain injury.
Metabolic syndrome is a constellation of metabolic and cardiovascular abnormalities including obesity, impaired glucose tolerance, dyslipidemia, and cardiovascular morbidity (Grundy et al., 2005). The adverse consequences of metabolic syndrome are typically linked to vascular disease, but associations with non-vascular diseases have been noted. Interestingly, children with epilepsy have a high rate of obesity at initial presentation (Daniels et al., 2009). It is unknown whether this association indicates a causal relationship between metabolic disease and seizure susceptibility.
Adiponectin is secreted by adipocytes and improves insulin sensitivity and fat oxidation (Ahima, 2006). Adiponectin is inversely correlated with adiposity, hence metabolic syndrome is associated with low plasma adiponectin (Ahima, 2006). While adiponectin deficiency has no apparent metabolic effects in lean mice, adiponectin deficiency in mice fed a high-fat diet (HFD) results in insulin resistance, hyperlipidemia, inflammation and vascular injury (Kubota et al., 2002; Ma et al., 2002; Maeda et al., 2002; Nawrocki et al., 2006). Low adiponectin levels are found in cerebrospinal fluid, and both adiponectin receptors, AdipoR1 and AdipoR2, are widely expressed in the brain (Yamauchi et al., 2003; Kusminski et al., 2007; Yamauchi et al., 2007). Adiponectin modulates hypothalamic and brainstem neuronal activity, and acts centrally to control peripheral metabolism (Qi et al., 2004; Fry et al., 2006; Hoyda et al., 2007; Kubota et al., 2007).
Adiponectin is protective against ischemic brain injury by modulating inflammatory pathways and endothelial function (Nishimura et al., 2008; Chen et al., 2009). Interestingly, PPARγ agonists, which are known to increase adiponectin expression, protect against seizure-related pathology (Maurois et al., 2008; Sun et al., 2008; Yu et al., 2008; Abdallah, 2010). Furthermore, the anti-epileptic drug valproic acid modulates PPARγ signaling, and alters adipoR1 and adiponectin expression (Qiao et al., 2006; Lan et al., 2008; Rauchenzauner et al., 2008). Adiponectin injected intracerebrally has also been shown to reduce kainic acid (KA)-induced excitotoxicity (Jeon et al., 2009). Thus, we hypothesized that adiponectin deficiency would enhance seizure sensitivity in the setting of metabolic syndrome. We fed C57BL/6J (wild type) and ADP-KO mice HFD or normal chow and compared body composition, glucose tolerance, lipids, KA-induced seizure, and hippocampal pathology. As predicted, adiponectin deficiency resulted in an increase in body fat, impaired glucose tolerance and increased lipids, and these changes were associated with increased seizure severity and hippocampal pathology.
C57BL/6J mice and ADP-KO mice bred on the same genetic background (n=17 per genotype), were fed HFD (Research Diets, New Brunswick, NJ, #D12451; 45% fat, 35% carbohydrate, 20% protein; 4.7 kcal/g) for 8-12 weeks (Takahashi et al., 2002). Control WT and adiponectin deficient mice (n=10 per genotype), were fed normal chow ( 5% fat, 49% carbohydrate, 24% protein; 4 kcal/g; LabDiet, Richmond, IN). Body composition was analyzed by nuclear magnetic resonance (NMR, Echo Medical Systems, Houston, TX) (Varela et al., 2008). To determine glucose tolerance, mice were fasted overnight (16 hours), tail glucose was measured (OneTouch Ultra glucometer, Johnson & Johnson), 2g/kg glucose was injected intraperitoneally (IP), and tail glucose was measured at 15, 30, 60 and 120 minutes. Serum collected at sacrifice were used to measure triglycerides, cholesterol and non-esterified fatty acids (NEFA) by enzymatic assay (Imai et al., 2007; Varela et al., 2008).
KA in saline was administered subcutaneously (20 mg/kg) or stereotaxically into the hippocampus (−1.8 mm, −1.8 mm, −1.8 mm relative to bregma, 100 ng). Subcutaneous saline was used as a control. Seizure activity was scored every 15 minutes for four hours using a modified Racine scale (0 normal, 1 hypoactivity, 2 rigidity, 3 rearing with repetitive head/forepaw movements, 4 rearing and falling, 5, continuous rearing/falling, 6 generalized convulsions) (McKhann et al., 2003).
Two days after peripheral KA, food was removed for 4 hours in the morning before sacrifice. Serum was obtained via cardiac puncture. Mice were perfused with phosphate buffered (PBS) followed by neutral buffered formalin. Brains were post-fixed overnight, embedded in paraffin and sectioned coronally (6 μm) for cresyl violet stain. Adjacent sections were subject to immunohistochemistry using the following antibodies: rat anti-GFAP (clone 2.2B10), rabbit anti-Iba1 (Wako Chemicals USA, Richmond, VA), rat anti-phospho-neurofilament (clone TA51), mouse anti-neurofilament (clone RMD020) and mouse anti-synaptophysin (clone SY38, Abcam, Cambridge, MA). The slides were scored by a neuropathologist on a scale of 1-4 (1 normal, 2 mild, 3 moderate, and 4 severe).
The effects of genotype and diet were assessed by unpaired t-test or ANOVA, and pair wise comparisons were analyzed with Fisher’s least significant difference test. For correlation analysis, seizure scores over time were used to calculate an area under the curve (AUC) followed by linear regressions between seizure AUC and metabolic parameters.
ADP-KO and WT mice were fed HFD to induce features of the metabolic syndrome, and assess its impact on KA-induced seizures. After two months on HFD, ADP-KO and WT mice had similar body weight (30.8+/− 0.8 g vs. 30.6 +/− 0.5 g; p=0.842, Fig 1A). However, ADP-KO mice had significantly greater fat mass (7.67 +/− 0.58 g vs. 4.63 +/− 0.27 g; p=0.0002) and less lean tissue mass (21.67 +/− 0.69 g vs. 24.66 +/− 0.35 g; p=0.0011) compared to WT mice (Fig 1A). We performed intraperitoneal glucose tolerance tests as a measure of glucose homeostasis. After overnight fasting, ADP-KO mice were hyperglycemic compared to WT mice (148.5 +/− 10.3 mg/dL vs. 115.6 +/− 11.7 mg/dL, p=0.049). After IP injection of glucose, blood glucose was higher in ADP-KO mice than WT (genotype p=0.0164, time p<0.0001, interaction p=0.2814; Fig 1B). At the time of sacrifice, ADP-KO mice had higher serum levels of triglycerides, NEFA and cholesterol than WT mice (Table 1). KA treatment decreased serum cholesterol levels, but this change was less compared to the effect of genotype (Table 1).
To determine whether features of metabolic syndrome resulting from adiponectin deficiency increased seizure severity, ADP-KO and WT mice were treated with a low dose of KA (20 mg/kg) and seizures were scored from 0 (no seizure) to 6 (tonic-clonic). ADP-KO mice were more sensitive to KA-induced seizure activity than WT, with peak seizure scores of 2.7 and 1.2, respectively (p=0.0079). Indeed, half of the ADP-KO mice had peak seizure scores of 3-4 while no WT mice scored higher than 2. Analysis of seizures over 4 hours showed that the mean score was higher for ADP-KO mice at all times (genotype p=0.0127, time p<0.0001, interaction p<0.0001; Fig 2A). The duration of seizures was longer in ADP-KO mice up to 120 minutes, compared to 30 minutes in WT mice. Thus, metabolic syndrome due to adiponectin deficiency resulted in more intense and prolonged seizure activity.
The downstream sequelae of seizures include gliosis, neurodegeneration, and neuronal reorganization (McKhann et al., 2003). The KA dose of 20 mg/kg is a low for C57BL/6J mice (Ferraro et al., 1995; McKhann et al., 2003), thus we predicted minimal gliosis and neurodegeneration in WT mice versus ADP-KO mice. Cresyl violet stained sections of brain and hippocampus showed no neurodegeneration in either WT or ADP-KO (Fig 3A), with the exception of one KA-treated ADP-KO mouse which showed severe loss of CA1 neurons (Fig 2D). Immunohistochemistry for glial fibrillary acidic protein (GFAP) showed mild astrocytosis in KA-treated WT mice relative to saline-treated mice, and considerably more astrocytosis in KA-treated ADP-KO mice (Fig 2B). Immunohistochemistry for Iba1 showed no microglial activation in WT mice and saline-treated ADP-KO mice. However, mild to moderate microglial activation was noted in ADP-KO mice, including microglial hypertrophy and clustering (Fig 2B). The single ADP-KO mouse with neurodegeneration showed profound glial activation (Fig 2D and data not shown). Semiquantitative image analysis demonstrated that KA treated ADP-KO mice showed significantly more astrocytic and microglial activation relative to WT mice (Fig 2C). Neurodegeneration was not statistically different between the two groups. Even when removing the one potential outlier with severe neurodegeneration, repeat analysis still indicated that ADP-KO mice showed significantly more glial pathology compared to WT mice (data not shown). Immunohistochemistry for synaptophysin or phosphorylated neurofilament did not show any evidence of synaptic sprouting or other structural changes (data not shown). These findings indicate that the worsening of seizure severity was accompanied by increased brain injury.
It is possible that altered body composition may change peripheral KA metabolism. To circumvent this issue, HFD-fed WT and ADP-KO mice were injected with a low KA dose (100 ng) directly into the hippocampus and examined after 2 weeks for chronic seizure related pathology. Severe neurodegeneration was evident in the hilum, CA3 and CA1 of ADP-KO, whereas neurodegeneration was mild or absent in WT (Fig 3A). Intrahippocampal KA also resulted in neuronal dispersion of the dentate gyrus, and enhanced synaptophysin immunostating in ADP-KO mice, suggesting extensive synaptic sprouting (Fig 3A). GFAP and Iba1 staining were increased indicative of reactive astrocytosis and microgliosis in ADP-KO mice (Figs 3A). Image analysis showed that ADP-KO displayed significantly increased granule cell dispersion (genotype p=0.0138, laterality p=0.0642, interaction p=0.0827), neurodegeneration (genotype p=0.0123, laterality p=0.0020, interaction p=0.0297), astrocytosis (genotype p=0.0185, laterality p=0.0030, interaction p=0.6679) and microgliosis (genotype p=0.0344, laterality p=0.0052, interaction p=0.5265) compared to WT (Fig 3B-E). Thus, adiponectin deficiency enhances seizure related pathology in response to peripheral or central KA treatment.
We hypothesized that an interaction between metabolic syndrome and adiponectin deficiency resulted in enhanced seizure activity. However, it was possible that adiponectin deficiency alone in the absence of metabolic changes may be sufficient to enhance seizure sensitivity. Thus, we examined the effects of KA in WT and ADP-KO mice fed normal chow diet. ADP-KO and WT mice had similar body weight, fat and lean mass, glucose tolerance and serum lipids (data not shown). Seizure activity was similar between WT and ADP-KO mice, peak seizure scores ranging from 0 to 1 (ADP-KO average peak score 0.6, WT average peak score 0.5, p=0.77). Temporal analysis of seizure scores showed no significant effect of genotype (genotype p=0.312, time p=0.121, interaction p=0.608).
Adiponectin deficiency is associated with increased adiposity in HFD mice, thus it is possible that seizure activity is associated with changes in metabolic parameters. We found a strong positive correlation between seizure severity and glucose intolerance (R2=0.5509, p=0.0057), cholesterol (R2=0.5341, p=0.0069), fat mass (R2=0.4391, p=0.0189) and NEFA (R2=0.4310, p=0.0282), and a negative correlation with lean mass (R2=0.4706, p=0.0138). In contrast, seizure severity was not associated with serum triglyceride (R2=0.1782, p=0.1717) or body weight (R2=0.0756, p=0.3869).
It is estimated that 5-10% of individuals develop non-febrile seizures or epilepsy in their lifetime (Hauser et al., 1993; Cockerell et al., 1995). Current efforts are underway to understand the co-morbid conditions associated with epilepsy. Associations between epilepsy and obesity are confounded by the known effects of anti-epileptic drugs on peripheral metabolism (Isojarvi et al., 1996). However, a recent study has shown an increase in obesity rates in children at initial presentation before the use of anti-epileptic agents (Daniels et al., 2009).
Adiponectin deficiency results in insulin resistance, glucose intolerance, dyslipidemia and vascular injury, characteristic of metabolic syndrome. These features are reversible by adiponectin treatment (Kubota et al., 2002; Ma et al., 2002; Maeda et al., 2002; Nawrocki et al., 2006). Moreover, adiponectin has been shown to ameliorate cerebrovascular injury in mice (Nishimura et al., 2008; Chen et al., 2009). We tested whether metabolic syndrome due to adiponectin deficiency would exacerbate seizure related brain injury. The KA dose in our study was below that required to induce seizures in C57BL/6J mice (Ferraro et al., 1995; McKhann et al., 2003), hence it is remarkable that clonic seizures (seizure score of 3+) occurred in 50% of HFD-fed ADP-KO mice. ADP-KO and WT mice on HFD had similar body weight and thus received the same KA doses. Furthermore, a low dose of intrahippocampal KA which normally causes mild brain pathology (Schauwecker, 2002), resulted in severe neuronal damage and gliosis in ADP-KO mice. Thus, the worsening of pathology in ADP-KO mice cannot be attributed to altered KA pharmacokinetics in ADP-KO mice.
Adiponectin deficiency in normal chow-fed mice did not increase seizure sensitivity. Rather, there was a trend towards higher seizure activity in HFD WT and ADP-KO mice relative to chow-fed counterparts. Thus, it appears that metabolic changes, rather than adiponectin, are the major determinant of seizure severity and hippocampal pathology. It is difficult to compare the effects of HFD versus normal chow diet on subcutaneous KA-induced seizure activity in HFD WT or ADP-KO mice, because the HFD mice were heavier and received a higher KA dose than chow-fed mice. Additional studies are needed to understand how adiponectin specifically alters metabolic parameters and seizures. Adiponectin receptors are distributed widely in the brain (Guillod-Maximin et al., 2009), and central adiponectin has potent electrophysiological effects (Fry et al., 2006; Hoyda et al., 2007), raising the possibility that adiponectin directly modifies seizure activity and brain pathology. Anti-epileptic drugs modulate various metabolic pathways (Isojarvi et al., 1996). For example, valproic acid regulates adiponectin and adipoR1 expression (Qiao et al., 2006; Rauchenzauner et al., 2008). PPARγ agonists including insulin sensitizing thiazolidinediones are protective in animal seizure models (Chen et al., 2009; Jeon et al., 2009), and also increase adiponectin levels (Nawrocki et al., 2006).
Other hormones associated with energy homeostasis influence seizures. Leptin and ghrelin inhibit seizures and protect against seizure-related neuropathology (Shanley et al., 2002; Obay et al., 2007; Erbayat-Altay et al., 2008; Guo et al., 2008; Obay et al., 2008; Xu et al., 2008; Lee et al., 2010; Obeid et al., 2010). These studies demonstrate that peripheral endocrine and metabolic factors are capable of modulating seizure threshold and seizure-related pathology by acting on CNS neurons to trigger intracellular signaling pathways or modulating neuronal activity. The results of the current study indicate that changes in metabolic parameters associated with adiponectin deficiency influence seizure activity and brain pathology. Understanding of the underlying mechanisms would provide a framework for prevention and treatment of epilepsy associated with metabolic syndrome.
This study was supported by NIH grants RO1-DK-062348 and PO1-DK-049210 (RSA) and T32-AG00255 and K08-AG039510 (EBL). We thank Drs. John Q. Trojanowski and Virginia M.-Y. Lee for providing antibody reagents, and the University of Pennsylvania Diabetes and Endocrinology Research Center (DERC) Mouse Phenotyping, Physiology and Metabolism Core for body composition analysis (NIH P30-DK19525).
Confilcts of interest: None