Transient anticonvulsant effects of time-restricted feeding in the 6-Hz mouse model.

INTRODUCTION: Intermittent fasting enhances neural bioenergetics, is neuroprotective, and elicits antioxidant effects in various animal models. There are conflicting findings on seizure protection, where intermittent fasting regimens often cause severe weight loss resembling starvation which is unsustainable long-term. Therefore, we tested whether a less intensive intermittent fasting regimen such as time-restricted feeding (TRF) may confer seizure protection. METHODS: Male CD1 mice were assigned to either ad libitum-fed control, continuous 8 h TRF, or 8 h TRF with weekend ad libitum food access (2:5 TRF) for one month. Body weight, food intake, and blood glucose levels were measured. Seizure thresholds were determined at various time points using 6-Hz and maximal electroshock seizure threshold (MEST) tests. Protein levels and mRNA expression of genes, enzyme activity related to glucose metabolism, as well as mitochondrial dynamics were assessed in the cortex and hippocampus. Markers of antioxidant defence were evaluated in the plasma, cortex, and liver. RESULTS: Body weight gain was similar in the ad libitum-fed and TRF mouse groups. In both TRF regimens, blood glucose levels did not change between the fed and fasted state and were higher during fasting than in the ad libitum-fed groups. Mice in the TRF group had increased seizure thresholds in the 6-Hz test on day 15 and on day 19 in a second cohort of 2:5 TRF mice, but similar seizure thresholds at other time points compared to ad libitum-fed mice. Continuous TRF did not alter MEST seizure thresholds on day 28. Mice in the TRF group showed increased maximal activity of pyruvate dehydrogenase in the cortex, which was accompanied by increased protein levels of mitochondrial pyruvate carrier 1 in the cortex and hippocampus. There were no other major changes in protein or mRNA levels associated with energy metabolism and mitochondrial dynamics in the brain, nor markers of antioxidant defence in the brain, liver, or plasma. CONCLUSIONS: Both continuous and 2:5 TRF regimens transiently increased seizure thresholds in the 6-Hz model at around 2 weeks, which coincided with stability of blood glucose levels during the fed and fasted periods. Our findings suggest that the lack of prolonged anticonvulsant effects in the acute electrical seizure models employed may be attributed to only modest metabolic and antioxidant adaptations found in the brain and liver. Our findings underscore the potential therapeutic value of TRF in managing seizure-related conditions.

Metabolic aspects of genetic ion channel epilepsies.

Nowadays, particularly in countries with high incomes, individual mutations in people affected by genetic epilepsies are identified, and genetic therapies are being developed. In addition, drugs are being screened to directly target specific mutations, and personalised medicine is possible. However, people with epilepsy do not yet benefit from these advances, and many types of epilepsies are medication-resistant, including Dravet syndrome. Thus, in the meantime, alternative and effective treatment options are needed. There is increasing evidence that metabolic deficits contribute to epileptic seizures and that such metabolic impairments may be amenable to treatment, with metabolic treatment options like the ketogenic diet being employed with some success. However, the brain metabolic alterations that occur in ion channel epilepsies are not well-understood, nor how these may differ from epilepsies that are of acquired and unknown origins. Here, we provide an overview of studies investigating metabolic alterations in epilepsies caused by mutations in the SCN1A and KCNA1 genes, which are currently the most studied ion channel epilepsies in animal models. The metabolic changes found in these models are likely to contribute to seizures. A metabolic basis of these ion channel epilepsies is supported by human and/or animal studies that show beneficial effects of the ketogenic diet, which may be mediated by the provision of auxiliary brain fuel in the form of ketone bodies. Other potentially more preferred dietary therapies including medium-chain triglycerides and triheptanoin have also been tested in a limited number of studies, but their efficacies remain to be clearly established. The extent to which brain metabolism is affected in people with Dravet syndrome, KCNA1 epilepsy and the models thereof still requires clarification. This requires more experiments that yield functional insight into metabolism.

ß-Hydroxybutyrate and Medium-Chain Fatty Acids are Metabolized by Different Cell Types in Mouse Cerebral Cortex Slices.

Ketogenic diets and medium-chain triglycerides are gaining attention as treatment of neurological disorders. Their major metabolites, ß-hydroxybutyrate (ßHB) and the medium-chain fatty acids (MCFAs) octanoic acid (C8) and decanoic acid (C10), are auxiliary brain fuels. To which extent these fuels compete for metabolism in different brain cell types is unknown. Here, we used acutely isolated mouse cerebral cortical slices to (1) compare metabolism of 200 µM [U-13C]C8, [U-13C]C10 and [U-13C]ßHB and (2) assess potential competition between metabolism of ßHB and MCFAs by quantifying metabolite 13C enrichment using gas chromatography-mass spectrometry (GC-MS) analysis. The 13C enrichment in most metabolites was similar with [U-13C]C8 and [U-13C]C10 as substrates, but several fold lower with [U-13C]ßHB. The 13C enrichment in glutamate was in a similar range for all three substrates, whereas the 13C enrichments in citrate and glutamine were markedly higher with both [U-13C]C8 and [U-13C]C10 compared with [U-13C]ßHB. As citrate and glutamine are indicators of astrocytic metabolism, the results indicate active MCFA metabolism in astrocytes, while ßHB is metabolized in a different cellular compartment. In competition experiments, 12C-ßHB altered 13C incorporation from [U-13C]C8 and [U-13C]C10 in only a few instances, while 12C-C8 and 12C-C10 only further decreased the low [U-13C]ßHB-derived 13C incorporation into citrate and glutamine, signifying little competition for oxidative metabolism between ßHB and the MCFAs. Overall, the data demonstrate that ßHB and MCFAs are supplementary fuels in different cellular compartments in the brain without notable competition. Thus, the use of medium-chain triglycerides in ketogenic diets is likely to be beneficial in conditions with carbon and energy shortages in both astrocytes and neurons, such as GLUT1 deficiency.

Vitamin B12 deficiency induces glucose intolerance, delays peak insulin levels and promotes ketogenesis in female rats.

Vitamin B12 (B12) deficiency is common among individuals with diabetes mellitus, but it is unknown if B12 deficiency contributes to impaired glucose homeostasis in this disorder. Female Sprague-Dawley rats were assigned to a control or B12-deficient diet for 4 weeks. Intraperitoneal glucose tolerance tests were performed after 25 days, and blood and liver samples were collected for metabolic profiling. B12 deficiency resulted in a prediabetic-like phenotype characterised by glucose intolerance, a delayed peak in plasma insulin levels following a glucose challenge and increased ketogenesis. We attributed increased ketogenesis to reduced liver anaplerosis, which limited the availability of the TCA cycle intermediates citrate, succinate and succinyl-CoA. This was associated with increased Mut mRNA levels and citrate synthase activity in the liver. One-carbon metabolite levels were altered in plasma and the liver, which was linked to reduced methylation capacity, altered amino acid levels and elevated Slc7a5 mRNA expression. Plasma folate and biotin levels were reduced, as were the majority of B vitamins in the liver. Changes in these B12-dependent processes and reduced B vitamin amounts likely contributed to deficits in glucose handling. Our findings highlight that B12 deficiency may promote the development of metabolic disorders like diabetes mellitus and emphasise the importance of adequate B12 intake for metabolic health.

Glyceryl triacetate feeding in mice increases plasma acetate levels but has no anticonvulsant effects in acute electrical seizure models.

INTRODUCTION: Acetate has been shown to have neuroprotective and anti-inflammatory effects. It is oxidized by astrocytes and can thus provide auxiliary energy to the brain in addition to glucose. Therefore, we hypothesized that it may protect against seizures, which is investigated here by feeding glyceryl triacetate (GTA), to provide high amounts of acetate without raising sodium or acid levels. METHOD: CD1 male mice were fed controlled diets with or without GTA for up to three weeks. Body weights, blood glucose levels, plasma short-chain fatty acid levels, and other hematological parameters were monitored. Seizure thresholds were determined in 6 Hz and maximal electroshock seizure threshold (MEST) tests. Antioxidant capacities were evaluated in the cerebral cortex and plasma using a ferric reducing antioxidant power (FRAP) assay and Trolox equivalent antioxidant capacity assay. RESULTS: Body weight gain was similar with both diets with and without GTA in two experiments. Glyceryl triacetate-fed groups showed 2-3- and 1.6-fold increased acetate and propionate levels in plasma, respectively. Glucose levels were unaltered in blood collected from the tail tip but increased in trunk blood. No differences were found in the activity of cerebral cortex acetyl-CoA synthetase. In the 6 Hz threshold test, seizure thresholds were lower by 3 mA and 2.4 mA after 8 and 14 days, respectively, in the GTA compared to the control diet-fed group, but showed no difference on day 16, showing that GTA has small, but inconsistent proconvulsant effects in this model. In MEST tests, a slightly increased seizure threshold (1 mA) was found on day 19 in the GTA-fed group, but not in another experiment on day 21. There were no differences in antioxidant capacity in plasma or cortex between the two groups. CONCLUSION: Glyceryl triacetate feeding showed no antioxidant effects nor beneficial changes in acute electrical seizure threshold mouse models, despite its ability to increase plasma acetate levels.

Brain glycogen content is increased in the acute and interictal chronic stages of the mouse pilocarpine model of epilepsy.

Glucose is the main brain fuel in fed conditions, while astrocytic glycogen is used as supplemental fuel when the brain is stimulated. Brain glycogen levels are decreased shortly after induced seizures in rodents, but little is known about how glycogen levels are affected interictally in chronic models of epilepsy. Reduced glutamine synthetase activity has been suggested to lead to increased brain glycogen levels in humans with chronic epilepsy. Here, we used the mouse pilocarpine model of epilepsy to investigate whether brain glycogen levels are altered, both acutely and in the chronic stage of the model. One day after pilocarpine-induced convulsive status epilepticus (CSE), glycogen levels were higher in the hippocampal formation, cerebral cortex, and cerebellum. Opposite to expected, this was accompanied by elevated glutamine synthetase activity in the hippocampus but not the cortex. Increased interictal glycogen amounts were seen in the hippocampal formation and cerebral cortex in the chronic stage of the model (21 days post-CSE), suggesting long-lasting alterations in glycogen metabolism. Glycogen solubility in the cerebral cortex was unaltered in this epilepsy mouse model. Glycogen synthase kinase 3 beta (Gsk3b) mRNA levels were reduced in the hippocampal formations of mice in the chronic stage, which may underlie the elevated brain glycogen content in this model. This is the first report of elevated interictal glycogen levels in a chronic epilepsy model. Increased glycogen amounts in the brain may influence seizure susceptibility in this model, and this warrants further investigation.

Fructose 1,6-bisphosphate is anticonvulsant and improves oxidative glucose metabolism within the hippocampus and liver in the chronic pilocarpine mouse epilepsy model.

Glucose metabolism is altered in epilepsy, and this may contribute to seizure generation. Recent research has shown that metabolic therapies including the ketogenic diet and medium chain triglycerides can improve energy metabolism in the brain. Fructose 1,6-bisphosphate (F16BP) is an intermediate of glycolysis and when administered exogenously is anticonvulsant in several rodent seizure models and may alter glucose metabolism. Here, we showed that F16BP elevated the seizure threshold in the acute 6-Hz mouse seizure model and investigated if F16BP could restore impairments in glucose metabolism occurring in the chronic stage of the pilocarpine mouse model of epilepsy. Two weeks after the pilocarpine injections, mice that experienced status epilepticus (SE, "epileptic") and did not experience SE (no SE, "nonepileptic") were injected with vehicle (0.9% saline) or F16BP (1 g/kg in 0.9% saline) daily for 5 consecutive days. At 3 weeks, mice were injected with [U-13C6]-glucose and the % enrichment of 13C in key metabolites in addition to the total levels of each metabolite was measured in the hippocampal formation and liver. Fructose 1,6-bisphosphate increased total GABA in the hippocampal formation, regardless of whether mice had experienced SE. In the hippocampal formation, F16BP prevented reductions in the % 13C enrichment of citrate, succinate, malate, glutamate, GABA and aspartate that occurred in the chronic stage of the pilocarpine model. Interestingly, % 13C enrichment in glucose-derived metabolites was reduced in the liver in the chronic stage of the pilocarpine model. Fructose 1,6-bisphosphate was also beneficial in the liver, preventing reductions in % 13C enrichment of lactate and alanine that were associated with SE. This study confirmed that F16BP is anticonvulsant and can improve elements of glucose metabolism that are dysregulated in the chronic stage of the pilocarpine model, which may be due to reduction of spontaneous seizures. Our results highlight that F16BP may be therapeutically beneficial for epilepsies refractory to treatment.

Maternal selenium deficiency in mice promotes sex-specific changes to urine flow and renal expression of mitochondrial proteins in adult offspring.

Selenium deficiency during pregnancy can impair fetal development and predispose offspring to thyroid dysfunction. Given that key selenoproteins are highly expressed in the kidney and that poor thyroid health can lead to kidney disease, it is likely that kidney function may be impaired in offspring of selenium-deficient mothers. This study utilized a mouse model of maternal selenium deficiency to investigate kidney protein glycation, mitochondrial adaptations, and urinary excretion in offspring. Female C57BL/6 mice were fed control (>190 µg selenium/kg) or low selenium (<50 µg selenium/kg) diets four weeks prior to mating, throughout gestation, and lactation. At postnatal day (PN) 170, offspring were placed in metabolic cages for 24 hr prior to tissue collection at PN180. Maternal selenium deficiency did not impact selenoprotein antioxidant activity, but increased advanced glycation end products in female kidneys. Male offspring had reduced renal Complex II and Complex IV protein levels and lower 24 hr urine flow. Although renal aquaporin 2 (Aqp2) and arginine vasopressin receptor 2 (Avpr2) mRNA were not altered by maternal selenium deficiency, a correlation between urine flow and plasma free T4 concentrations in male but not female offspring suggests that programed thyroid dysfunction may be mediating impaired urine flow. This study demonstrates that maternal selenium deficiency can lead to long-term deficits in kidney parameters that may be secondary to impaired thyroid dysfunction. Considering the significant burden of renal dysfunction as a comorbidity to metabolic diseases, improving maternal selenium intake in pregnancy may be one simple measure to prevent lifelong disease.