Adiponectin deficiency increases body fat, glucose and lipids
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, ). 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 (). 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; ). At the time of sacrifice, ADP-KO mice had higher serum levels of triglycerides, NEFA and cholesterol than WT mice (). KA treatment decreased serum cholesterol levels, but this change was less compared to the effect of genotype ().
Effects of HFD on body composition and glucose tolerance
Effects of adiponectin deficiency and kainic acid treatment on serum lipids
Adiponectin deficiency increases seizure severity
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; ). 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.
Kainic-acid induced seizure in mice fed HFD
Adiponectin deficiency increases post-seizure hippocampal pathology
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 (), with the exception of one KA-treated ADP-KO mouse which showed severe loss of CA1 neurons (). 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 (). 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 (). The single ADP-KO mouse with neurodegeneration showed profound glial activation ( 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 (). 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.
Intrahippocampal kainic-acid and seizure-related pathology
Adiponectin deficiency increases chronic seizure related pathology
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 (). Intrahippocampal KA also resulted in neuronal dispersion of the dentate gyrus, and enhanced synaptophysin immunostating in ADP-KO mice, suggesting extensive synaptic sprouting (). GFAP and Iba1 staining were increased indicative of reactive astrocytosis and microgliosis in ADP-KO mice (). 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 (). Thus, adiponectin deficiency enhances seizure related pathology in response to peripheral or central KA treatment.
Adiponectin deficiency in the absence of metabolic syndrome does not alter seizure activity
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).