Potential mechanisms to modify impaired glucose metabolism in neurodegenerative disorders.

Neurodegeneration refers to the selective and progressive loss-of-function and atrophy of neurons, and is present in disorders such as Alzheimer's, Huntington's, and Parkinson's disease. Although each disease presents with a unique pattern of neurodegeneration, and subsequent disease phenotype, increasing evidence implicates alterations in energy usage as a shared and core feature in the onset and progression of these disorders. Indeed, disturbances in energy metabolism may contribute to the vulnerability of neurons to apoptosis. In this review we will outline these disturbances in glucose metabolism, and how fatty acids are able to compensate for this impairment in energy production in neurodegenerative disorders. We will also highlight underlying mechanisms that could contribute to these alterations in energy metabolism. A greater understanding of these metabolism-neurodegeneration processes could lead to improved treatment options for neurodegenerative disease patients.

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.

Glucose clearance and uptake is increased in the SOD1G93A mouse model of amyotrophic lateral sclerosis through an insulin-independent mechanism.

Metabolic disturbances are associated with the progression of the neurodegenerative disorder, amyotrophic lateral sclerosis (ALS). However, the molecular events that drive energy imbalances in ALS are not completely understood. In this study, we aimed to elucidate deficits in energy homeostasis in the SOD1G93A mouse model of ALS. SOD1G93A mice and their wild-type littermates underwent indirect calorimetry and intraperitoneal glucose/insulin tolerance tests at both the onset and mid-symptomatic stages of the disease. Glucose uptake and the plasma glucoregulatory hormone profiles were analyzed. Pancreatic islet cell mass and function were assessed by measuring hormone concentrations and secretion in isolated islets, and pancreatic α- and ß-cell immunoreactive areas. Finally, we profiled liver glycogen metabolism by measuring glucagon concentrations and liver metabolic gene expressions. We identified that mid-symptomatic SOD1G93A mice have increased oxygen consumption and faster exogenous glucose uptake, despite presenting with normal insulin tolerance. The capacity for pancreatic islets to secrete insulin appears intact, however, islet cell insulin concentrations and ß-cell mass were reduced. Fasting glucose homeostasis was also disturbed, along with increased liver glycogen stores, despite elevated circulating glucagon, suggesting that glucagon signaling is impaired. Metabolic gene expression profiling of livers indicated that glucose cannot be utilized efficiently in SOD1G93A mice. Overall, we demonstrate that glucose homeostasis and uptake are altered in SOD1G93A mice, which is linked to an increase in insulin-independent glucose uptake, and a loss of ß-cells, insulin production, and glucagon sensitivity. This suggests that the hormonal regulation of glucose concentrations may contribute to the progression of disease in this ALS mouse model.

Absence of Receptor for Advanced Glycation End Product (RAGE) Reduces Inflammation and Extends Survival in the hSOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron degenerative disease that is without effective treatment. The receptor for advanced glycation end products (RAGE) is a major component of the innate immune system that has been implicated in ALS pathogenesis. However, the contribution of RAGE signalling to the neuroinflammation that underlies ALS neurodegeneration remains unknown. The present study therefore generated SOD1G93A mice lacking RAGE and compared them with SOD1G93A transgenic ALS mice in respect to disease progression (i.e. body weight, survival and muscle strength), neuroinflammation and denervation markers in the spinal cord and tibialis anterior muscle. We found that complete absence of RAGE signalling exerted a protective effect on SOD1G93A pathology, slowing disease progression and significantly extending survival by ~ 3 weeks and improving motor function (rotarod and grip strength). This was associated with reduced microgliosis, cytokines, innate immune factors (complement, TLRs, inflammasomes), and oxidative stress in the spinal cord, and a reduction of denervation markers in the tibialis anterior muscle. We also documented that RAGE mRNA expression was significantly increased in the spinal cord and muscles of preclinical SOD1 and TDP43 models of ALS, supporting a widespread involvement for RAGE in ALS pathology. In summary, our results indicate that RAGE signalling drives neuroinflammation and contributes to neurodegeneration in ALS and highlights RAGE as a potential immune therapeutic target for ALS.

The potential interplay between energy metabolism and innate complement activation in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron disease without effective treatment. Although the precise mechanisms leading to ALS are yet to be determined, there is now increasing evidence implicating the defective energy metabolism and components of the innate immune complement system in the onset and progression of its motor phenotypes. This review will survey the mechanisms by which the energy metabolism and the complement system are altered during the disease progression of ALS and how it can contribute to disease. Furthermore, it will also examine how complement activation can modify the energy metabolism in metabolic disorders, in order to highlight how the complement system and energy metabolism may be linked in ALS.

The effect of dichloroacetate in mouse models of epilepsy.

A recent report has found that glucose oxidation and the activity of pyruvate dehydrogenase (PDH) are reduced in the chronic stage of the pilocarpine mouse epilepsy model. This is likely caused by increased phosphorylation by PDH kinase of the E1α subunit of PDH, downregulating its activity. Inhibition of this phosphorylation has not yet been explored as a possible approach to treat epilepsy. Chronic dichloroacetate (DCA, 50 and 100 mg/kg/day) treatment was tested in acute seizure and the chronic pilocarpine models. We also determined the effects on phosphorylation state, activity and protein levels of PDH in the chronic stage of the pilocarpine model. DCA treatment did not increase latencies to seizures in the acute flurothyl seizure test and was slightly proconvulsant in the 6 Hz test. The latencies to seizures in a second-hit flurothyl test were decreased in SE vs. No SE mice in the chronic stage, but were not restored by DCA. In mice that had experienced pilocarpine-induced SE and were in the chronic "epileptic" stage of the model, PDH activity was reduced by 65% compared to "healthy" No SE mice. This was partially alleviated with DCA treatment. Also, PDH protein levels were decreased by 37% and phosphorylation at Ser300 of PDH was increased by 52% in SE mice, but were not significantly changed with DCA. Moreover DCA treatment decreased the amounts of total PDH by 23% in No SE mice, which may explain the proconvulsant effects in the 6 Hz test. The reduction in PDH protein levels during the chronic epileptic stage suggests increased degradation of the protein, which may contribute to the deficient glucose oxidation found in epilepsy. Taken together, DCA did not have any anti-convulsant effects in the tested models. Future studies utilising other PDH kinase inhibitors are required to determine whether this treatment approach is viable.

Impaired hippocampal glucose metabolism during and after flurothyl-induced seizures in mice: Reduced phosphorylation coincides with reduced activity of pyruvate dehydrogenase.

OBJECTIVE: To determine changes in glucose metabolism and the enzymes involved in the hippocampus ictally and postictally in the acute mouse flurothyl seizure model. METHODS: [U-13 C]-Glucose was injected (i.p.) prior to, or following a 5 min flurothyl-induced seizure. Fifteen minutes later, mice were killed and the total metabolite levels and % 13 C enrichment were analyzed in the hippocampal formation using gas chromatography-mass spectrometry. Activities of key metabolic and antioxidant enzymes and the phosphorylation status of pyruvate dehydrogenase were measured, along with lipid peroxidation. RESULTS: During seizures, total lactate levels increased 1.7-fold; however, [M + 3] enrichment of both lactate and alanine were reduced by 30% and 43%, respectively, along with a 28% decrease in phosphofructokinase activity. Postictally the % 13 C enrichments of all measured tricarboxylic acid (TCA) cycle intermediates and the amino acids were reduced by 46-93%. At this time, pyruvate dehydrogenase (PDH) activity was 56% of that measured in controls, and there was a 1.9-fold increase in the phosphorylation of PDH at ser232. Phosphorylation of PDH is known to decrease its activity. SIGNIFICANCE: Here, we show that the increase of lactate levels during flurothyl seizures is from a source other than [U-13 C]-glucose, such as glycogen. Surprisingly, although we saw a reduction in phosphofructokinase activity during the seizure, metabolism of [U-13 C]-glucose into the TCA cycle seemed unaffected. Similar to our recent findings in the chronic phase of the pilocarpine model, postictally the metabolism of glucose by glycolysis and the TCA cycle was impaired along with reduced PDH activity. Although this decrease in activity may be a protective mechanism to reduce oxidative stress, which is observed in the flurothyl model, ATP is critical to the recovery of ion and neurotransmitter balance and return to normal brain function. Thus we identified promising novel strategies to enhance energy metabolism and recovery from seizures.

Alterations in Cytosolic and Mitochondrial [U-13C]Glucose Metabolism in a Chronic Epilepsy Mouse Model.

Temporal lobe epilepsy is a common form of adult epilepsy and shows high resistance to treatment. Increasing evidence has suggested that metabolic dysfunction contributes to the development of seizures, with previous studies indicating impairments in brain glucose metabolism. Here we aim to elucidate which pathways involved in glucose metabolism are impaired, by tracing the hippocampal metabolism of injected [U-13C]glucose (i.p.) during the chronic stage of the pilocarpine-status epilepticus mouse model of epilepsy. The enrichment of 13C in the intermediates of glycolysis and the TCA cycle were quantified in hippocampal extracts using liquid chromatography-tandem mass spectroscopy, along with the measurement of the activities of enzymes in each pathway. We show that there is reduced incorporation of 13C in the intermediates of glycolysis, with the percentage enrichment of all downstream intermediates being highly correlated with those of glucose 6-phosphate. Furthermore, the activities of all enzymes in this pathway including hexokinase and phosphofructokinase were unaltered, suggesting that glucose uptake is reduced in this model without further impairments in glycolysis itself. The key findings were 33% and 55% losses in the activities of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, respectively, along with reduced 13C enrichment in TCA cycle intermediates. This lower 13C enrichment is best explained in part by the reduced enrichment in glycolytic intermediates, whereas the reduction of key TCA cycle enzyme activity indicates that TCA cycling is also impaired in the hippocampal formation. Together, these data suggest that multitarget approaches may be necessary to restore metabolism in the epileptic brain.

Tridecanoin is anticonvulsant, antioxidant, and improves mitochondrial function.

The hypothesis that chronic feeding of the triglycerides of octanoate (trioctanoin) and decanoate (tridecanoin) in "a regular non-ketogenic diet" is anticonvulsant was tested and possible mechanisms of actions were subsequently investigated. Chronic feeding of 35E% of calories from tridecanoin, but not trioctanoin, was reproducibly anticonvulsant in two acute CD1 mouse seizure models. The levels of beta-hydroxybutyrate in plasma and brain were not significantly increased by either treatment relative to control diet. The respective decanoate and octanoate levels are 76 µM and 33 µM in plasma and 1.17 and 2.88 nmol/g in brain. Tridecanoin treatment did not alter the maximal activities of several glycolytic enzymes, suggesting that there is no reduction in glycolysis contributing to anticonvulsant effects. In cultured astrocytes, 200 µM of octanoic and decanoic acids increased basal respiration and ATP turnover, suggesting that both medium chain fatty acids are used as fuel. Only decanoic acid increased mitochondrial proton leak which may reduce oxidative stress. In mitochondria isolated from hippocampal formations, tridecanoin increased respiration linked to ATP synthesis, indicating that mitochondrial metabolic functions are improved. In addition, tridecanoin increased the plasma antioxidant capacity and hippocampal mRNA levels of heme oxygenase 1, and FoxO1.

Alternative Fuels in Epilepsy and Amyotrophic Lateral Sclerosis.

This review summarises the recent findings on metabolic treatments for epilepsy and Amyotrophic Lateral Sclerosis (ALS) in honour of Professor Ursula Sonnewald. The metabolic impairments in rodent models of these disorders as well as affected patients are being discussed. In both epilepsy and ALS, there are defects in glucose uptake and reduced tricarboxylic acid (TCA) cycling, at least in part due to reduced amounts of C4 TCA cycle intermediates. In addition there are impairments in glycolysis in ALS. A reduction in glucose uptake can be addressed by providing the brain with alternative fuels, such as ketones or medium-chain triglycerides. As anaplerotic fuels, such as the triglyceride of heptanoate, triheptanoin, refill the TCA cycle C4/C5 intermediate pool that is deficient, they are ideal to boost TCA cycling and thus the oxidative metabolism of all fuels.

Triheptanoin Protects Motor Neurons and Delays the Onset of Motor Symptoms in a Mouse Model of Amyotrophic Lateral Sclerosis.

There is increasing evidence that energy metabolism is disturbed in Amyotrophic Lateral Sclerosis (ALS) patients and animal models. Treatment with triheptanoin, the triglyceride of heptanoate, is a promising approach to provide alternative fuel to improve oxidative phosphorylation and aid ATP generation. Heptanoate can be metabolized to propionyl-CoA, which after carboxylation can produce succinyl-CoA and thereby re-fill the tricarboxylic acid (TCA) cycle (anaplerosis). Here we tested the hypothesis that treatment with triheptanoin prevents motor neuron loss and delays the onset of disease symptoms in female mice overexpressing the mutant human SOD1G93A (hSOD1G93A) gene. When oral triheptanoin (35% of caloric content) was initiated at P35, motor neuron loss at 70 days of age was attenuated by 33%. In untreated hSOD1G93A mice, the loss of hind limb grip strength began at 16.7 weeks. Triheptanoin maintained hind limb grip strength for 2.8 weeks longer (p<0.01). Loss of balance on the rotarod and reduction of body weight were delayed by 13 and 11 days respectively (both p<0.01). Improved motor function occurred in parallel with alterations in the expression of genes associated with muscle metabolism. In gastrocnemius muscles, the mRNA levels of pyruvate, 2-oxoglutarate and succinate dehydrogenases and methyl-malonyl mutase were reduced by 24-33% in 10 week old hSOD1G93A mice when compared to wild-type mice, suggesting that TCA cycling in skeletal muscle may be slowed in this ALS mouse model at a stage when muscle strength is still normal. At 25 weeks of age, mRNA levels of succinate dehydrogenases, glutamic pyruvic transaminase 2 and the propionyl carboxylase ß subunit were reduced by 69-84% in control, but not in triheptanoin treated hSOD1G93A animals. Taken together, our results suggest that triheptanoin slows motor neuron loss and the onset of motor symptoms in ALS mice by improving TCA cycling.

Triheptanoin partially restores levels of tricarboxylic acid cycle intermediates in the mouse pilocarpine model of epilepsy.

Triheptanoin, the triglyceride of heptanoate, is anticonvulsant in various epilepsy models. It is thought to improve energy metabolism in the epileptic brain by re-filling the tricarboxylic acid (TCA) cycle with C4-intermediates (anaplerosis). Here, we injected mice with [1,2-(13) C]glucose 3.5-4 weeks after pilocarpine-induced status epilepticus (SE) fed either a control or triheptanoin diet. Amounts of metabolites and incorporations of (13) C were determined in extracts of cerebral cortices and hippocampal formation and enzyme activity and mRNA expression were quantified. The percentage enrichment with two (13) C atoms in malate, citrate, succinate, and GABA was reduced in hippocampal formation of control-fed SE compared with control mice. Except for succinate, these reductions were not found in triheptanoin-fed SE mice, indicating that triheptanoin prevented a decrease of TCA cycle capacity. Compared to those on control diet, triheptanoin-fed SE mice showed few changes in most other metabolite levels and their (13) C labeling. Reduced pyruvate carboxylase mRNA and enzyme activity in forebrains and decreased [2,3-(13) C]aspartate amounts in cortex suggest a pyruvate carboxylation independent source of C-4 TCA cycle intermediates. Most likely anaplerosis was kept unchanged by carboxylation of propionyl-CoA derived from heptanoate. Further studies are proposed to fully understand triheptanoin's effects on neuroglial metabolism and interaction.

Alterations of hippocampal glucose metabolism by even versus uneven medium chain triglycerides.

Medium chain triglycerides (MCTs) are used to treat neurologic disorders with metabolic impairments, including childhood epilepsy and early Alzheimer's disease. However, the metabolic effects of MCTs in the brain are still unclear. Here, we studied the effects of feeding even and uneven MCTs on brain glucose metabolism in the mouse. Adult mice were fed 35% (calories) of trioctanoin or triheptanoin (the triglycerides of octanoate or heptanoate, respectively) or a matching control diet for 3 weeks. Enzymatic assays and targeted metabolomics by liquid chromatography tandem mass spectrometry were used to quantify metabolites in extracts from the hippocampal formations (HFs). Both oils increased the levels of ß-hydroxybutyrate, but no other significant metabolic alterations were observed after triheptanoin feeding. The levels of glucose 6-phosphate and fructose 6-phosphate were increased in the HF of mice fed trioctanoin, whereas levels of metabolites further downstream in the glycolytic pathway and the pentose phosphate pathway were reduced. This indicates that trioctanoin reduces glucose utilization because of a decrease in phosphofructokinase activity. Trioctanoin and triheptanoin showed similar anticonvulsant effects in the 6 Hz seizure model, but it remains unknown to what extent the anticonvulsant mechanism(s) are shared. In conclusion, triheptanoin unlike trioctanoin appears to not alter glucose metabolism in the healthy brain.

Brain mitochondrial metabolic dysfunction and glutamate level reduction in the pilocarpine model of temporal lobe epilepsy in mice.

Although certain metabolic characteristics such as interictal glucose hypometabolism are well established for temporal lobe epilepsy (TLE), its pathogenesis still remains unclear. Here, we performed a comprehensive study of brain metabolism in a mouse model of TLE, induced by pilocarpine-status epilepticus (SE). To investigate glucose metabolism, we injected mice 3.5-4 weeks after SE with [1,2-(13)C]glucose before microwave fixation of the head. Using (1)H and (13)C nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry and high-pressure liquid chromatography, we quantified metabolites and (13)C labeling in extracts of cortex and hippocampal formation (HF). Hippocampal levels of glutamate, glutathione and alanine were decreased in pilocarpine-SE mice compared with controls. Moreover, the contents of N-acetyl aspartate, succinate and reduced nicotinamide adenine dinucleotide (phosphate) NAD(P)H were decreased in HF indicating impairment of mitochondrial function. In addition, the reduction in (13)C enrichment of hippocampal citrate and malate suggests decreased tricarboxylic acid (TCA) cycle turnover in this region. In cortex, we found reduced (13)C labeling of glutamate, glutamine and aspartate via the pyruvate carboxylation and pyruvate dehydrogenation pathways, suggesting slower turnover of these amino acids and/or the TCA cycle. In conclusion, mitochondrial metabolic dysfunction and altered amino-acid metabolism is found in both cortex and HF in this epilepsy model.