Wednesday, 11 July 2018

Ketones and Autism Part 1 - Ketones, Epilepsy, GABA and Gut Bacteria

Today’s post is the first in a short series about ketones, that looks into very specific areas of the science.
There was an earlier post on the Ketogenic Diet (KD).

The Clever Ketogenic Diet for some Autism

We already know, anecdotally, that some people with autism respond well to the Ketogenic Diet (KD) and some to just ketone supplements. We also know from some very small clinical trials that a minority of those with autism benefit from the KD. 

What percentage of people with autism respond to ketones?
This is the important question and the simple answer appears to be a minority, albeit a significant minority. A lot depends on what you mean by autism and what you judge to be a response. From reading up on the subject I would estimate that about 20% respond well, but which 20%?

Why do some people with autism respond to ketones and how do you maximize the effect?
There is quite a lot of useful information in the literature, but it is clear that most people do not respond to ketones, so you really need to know if your case is one of the minority that do respond, before getting too carried away with changing diet. From research on people wanting to lose weight there are plenty of practical tips to maximize ketones, even drinking coffee produces more ketones; increasing the hours you fast each day also helps. 
If you have a healthy, sporty, child with autism you might want more ketones but you do not want to lose weight.

Do you have a responder? 
There does not yet appear to be a way to predict who will respond well to ketones, other than those people who have seizures. 
You can read all about ketones or just try them out. To try them out, a good place to start is with the exogenous ketone supplements mainly sold to people trying to lose weight. It looks like 10ml of C8 oil and 10ml of BHB ester is a good place to start and soon you may produce enough ketones to establish if someone is a responder, then you just have to figure out why they are a responder and then to maximize this effect, which might involve things other than ketone supplements.  
You can measure ketones in urine and so you can see whether you have reached the level found in trials that produces a positive effect in some people. If you can establish that you have indeed produced this level of ketones and you see no effect, then it is time to cross ketones and the ketogenic diet off your to-do list.  
If you are fortunate to find a responder, then you can move forward with trying to maximize the benefit. 

Ketone supplements
Ketone supplements can be extremely expensive when you use the recommended dosage and may contain quite large amounts of things that you might not want (sodium, potassium and calcium for example).  Always read the labels.
It is clear that some of these products are much more effective at producing ketones than others. For a change, you can measure their effectiveness and avoid wasting your money. Ketone testing strips are inexpensive. 

Ketone Posts 

·        Part1 - Ketones, epilepsy, GABA and gut bacteria

·        Ketones as a fuel, Alzheimer’s and GLUT1 deficiency

·        Ketones and microglia

·        Ketones as HDAC inhibitors and epigenetic modifiers

·        Ketones, exercise and BDNF

·        Maximizing ketones through diet, fasting, exercise, coffee and supplementation

Ketones, epilepsy, GABA and gut bacteria
Today’s post just looks at the effect of ketones on the neurotransmitter GABA, which should be inhibitory, but in much autism is actually excitatory. The GABA switch failed to flip just after birth and neurons remain in an immature state, due to too many NKCC1 chloride transporters and too few KCC2 chloride transporters. With too much chloride inside neurons GABA has an excitatory effect on neurons causing them to fire when they should not.
In epilepsy, too much excitation from Glutamate and too little inhibition from GABA may lead to seizures. So, in some types of epilepsy you want to increase GABA and reduce Glutamate, i.e. you want to increase the GABA/Glutamate ratio.
In autism it is not clear that increasing the GABA/Glutamate ratio is going to help, it all depends on the kind of autism. Much of this blog is about changing the effect of GABA (flipping the GABA switch) and not changing the amount of it.
In some people with autism and epilepsy ketones resolve the seizures but do not improve the autism.
In other people with autism and no seizures, ketones improve their autism. This may, or may not, be due to the effect ketones have on GABA.  
Two issues are looked into in this post: -
·        Do ketones affect NKCC1/KCC2 expression and hence the effect of GABA in the brain?

·        Do ketones affect the amount of GABA and Glutamate in key parts of the brain?
The good news is that research into epilepsy shows that ketones do indeed have an effect on GABA, but it is highly disputed whether they modify NKCC1/KCC2 expression and the GABA switch from immature to mature neurons. 

The KD and Epilepsy
The KD has been used to treat epilepsy for almost a century, but until very recently nobody really knew why it worked.
A recent very thoughtful study at UCLA has shown that the KD mediates its anti-seizure effects via changes to the bacteria in the gut. The researchers identified the two bacteria and then showed that, at least in mice, the same anti-seizure effect provided by the KD could be provided just by adding these two bacteria (i.e. no need to follow the ketogenic diet).

UCLA scientists have identified specific gut bacteria that play an essential role in the anti-seizure effects of the high-fat, low-carbohydrate ketogenic diet. The study, published today in the journal Cell, is the first to establish a causal link between seizure susceptibility and the gut microbiota — the 100 trillion or so bacteria and other microbes that reside in the human body’s intestines.

The ketogenic diet has numerous health benefits, including fewer seizures for children with epilepsy who do not respond to anti-epileptic medications, said Elaine Hsiao, UCLA assistant professor of integrative biology and physiology in the UCLA College, and senior author of the study. However, there has been no clear explanation for exactly how the diet aids children with epilepsy.
Researchers in Hsiao’s laboratory hypothesized that the gut microbiota is altered through the ketogenic diet and is important for the diet’s anti-seizure effects. Hsiao’s research team conducted a comprehensive investigation into whether the microbiota influences the ability of the diet to protect against seizures and if so, how the microbiota achieves these effects.
In a study of mice as a model to more thoroughly understand epilepsy, the researchers found that the diet substantially altered the gut microbiota in fewer than four days, and mice on the diet had significantly fewer seizures.
To test whether the microbiota is important for protection against seizures, the researchers analyzed the effects of the ketogenic diet on two types of mice: those reared as germ-free in a sterile laboratory environment and mice treated with antibiotics to deplete gut microbes.
“In both cases, we found the ketogenic diet was no longer effective in protecting against seizures,” said lead author Christine Olson, a UCLA graduate student in Hsiao’s laboratory. “This suggests that the gut microbiota is required for the diet to effectively reduce seizures.”
The biologists identified the precise order of organic molecules known as nucleotides from the DNA of gut microbiota to determine which bacteria were present and at what levels after the diet was administered. They identified two types of bacteria that were elevated by the diet and play a key role in providing this protection: Akkermansia muciniphila and Parabacteroides species.
With this new knowledge, they studied germ-free mice that were given these bacteria.
“We found we could restore seizure protection if we gave these particular types of bacteria together,” Olson said. “If we gave either species alone, the bacteria did not protect against seizures; this suggests that these different bacteria perform a unique function when they are together.”
The researchers measured levels of hundreds of biochemicals in the gut, blood and hippocampus, a region of the brain that plays an important role in spreading seizures in the brain. They found that the bacteria that were elevated by the ketogenic diet alter levels of biochemicals in the gut and the blood in ways that affect neurotransmitters in the hippocampus.
How do the bacteria do this? “The bacteria increased brain levels of GABA — a neurotransmitter that silences neurons — relative to brain levels of glutamate, a neurotransmitter that activates neurons to fire,” said co-author Helen Vuong, a postdoctoral scholar in Hsiao’s laboratory.
“This study inspires us to study whether similar roles for gut microbes are seen in people that are on the ketogenic diet,” Vuong said.
“The implications for health and disease are promising, but much more research needs to be done to test whether discoveries in mice also apply to humans,” said Hsiao, who is also an assistant professor of medicine in the David Geffen School of Medicine at UCLA.
On behalf of the Regents of the University of California, the UCLA Technology Development Group has filed a patent on Hsiao’s technology that mimics the ketogenic diet to provide seizure protection. It has exclusively licensed it to a start-up company Hsiao has helped to launch that will examine the potential clinical applications of her laboratory’s findings.
Here is Hsiao’s new start-up:

“We are hacking the ketogenic diet to identify microbes that have therapeutic potential for the treatment of epilepsy,” says Bloom CEO Tony Colasin.
The San Diego-based start-up isn’t announcing how much seed funding it raised, but Colasin plans to move fast and get a product on the market in a mere two to three years. That’s because Bloom will first develop a medical food based on the two kinds of bacteria identified in Hsiao’s study. Bloom also has plans to optimize the bacterial strains for specific kinds of epilepsy to develop a traditional approved drug.

The way the bacteria help control seizures actually appears to be similar to the mechanism of many commercial ant epilepsy drugs. The microbes’ metabolism increases the ratio of inhibitory to excitatory neurotransmitters in the brain—specifically, higher levels of gamma-aminobutyric acid (GABA) relative to levels of glutamate.
Bloom is also considering how the microbiome benefits of the ketogenic diet could be helpful in other neurological conditions, including autism, depression, and Parkinson’s disease. “It is early days, but we are excited about the potential,” Colasin says.

Here is the full paper: -

The ketogenic diet (KD) is used to treat refractory epilepsy, but the mechanisms underlying its neuroprotective effects remain unclear. Here, we show that the gut microbiota is altered by the KD and required for protection against acute electrically induced seizures and spontaneous tonic-clonic seizures in two mouse models. Mice treated with antibiotics or reared germ free are resistant to KD-mediated seizure protection. Enrichment of, and gnotobiotic co-colonization with, KD-associated Akkermansia and Parabacteroides restores seizure protection. Moreover, transplantation of the KD gut microbiota and treatment with Akkermansia and Parabacteroides each confer seizure protection to mice fed a control diet. Alterations in colonic lumenal, serum, and hippocampal metabolomic profiles correlate with seizure protection, including reductions in systemic gamma-glutamylated amino acids and elevated hippocampal GABA/glutamate levels. Bacterial cross-feeding decreases gamma-glutamyltranspeptidase activity, and inhibiting gamma-glutamylation promotes seizure protection in vivo. Overall, this study reveals that the gut microbiota modulates host metabolism and seizure susceptibility in mice.

Achieving the Gut Bacteria changes of the KD without the diet

Since the ketogenic diet (KD) is very restrictive, it would be much more convenient to achieve the GABA/Glutamate effect in a simpler way. You cannot currently just buy these two bacteria as a supplement.  There would seem to be 2 other obvious options: -

·        Take exogenous ketone supplements and hope this causes the same gut bacterial changes produced by the KD. No evidence exists.

·        Use other known methods to increase to increase Akkermansia muciniphila and Parabacteroides species in a similar way to that likely being developed by Bloom Sciences in San Diego. Bloom are developing a medical food to achieve this, so it will require a prescription and it will be costly. 

Increasing Akkermansia muciniphila can be achieved using fructooligosaccharides (FOS). FOS is included in many types of formula milk for babies and is sold as a supplement.
Metformin, a drug used to treat type 2 diabetes, greatly increases Akkermansia muciniphila. Vancomycin, the antibiotic that stays in the gut, also greatly increases Akkermansia muciniphila, but it is also going to wipe out many bacteria.
The research shows you also need the second bacteria Parabacteroides, of which there are many types. These bacteria are found in high levels in people following a Mediterranean type diet but it can be increased using Resistant Starch Type 4. This type of starch has been chemically modified to resist digestion. This starch is sold as food ingredient to add to bakery products.

Viable A. muciniphila and fructooligosaccharides contently promote A. muciniphila. 
Metformin and vancomycin also significantly promote A. muciniphila.

“Akkermansia can also be increased by consuming polyphenol-rich foods, including:

·         pomegranate (attributed to ellagitannins and their metabolites)

·      grape polyphenols (grape seed extract) (proanthocyanidin-rich extracts may increase mucus secretion, therefore creating a favorable environment for Akkermansia to thrive) 

·   cranberries “ 

The abundance of Parabacteroides distasonis (P = .025) and Faecalibacterium prausnitzii (P = .020) increased after long-term consumption of the Med diet and the LFHCC diet, respectively.

Resistant starches types 2 and 4 have differential effects on the composition of the fecal microbiota in human subjects.



To systematically develop dietary strategies based on resistant starch (RS) that modulate the human gut microbiome, detailed in vivo studies that evaluate the effects of different forms of RS on the community structure and population dynamics of the gut microbiota are necessary. The aim of the present study was to gain a community wide perspective of the effects of RS types 2 (RS2) and 4 (RS4) on the fecal microbiota in human individuals.


Ten human subjects consumed crackers for three weeks each containing either RS2, RS4, or native starch in a double-blind, crossover design. Multiplex sequencing of 16S rRNA tags revealed that both types of RS induced several significant compositional alterations in the fecal microbial populations, with differential effects on community structure. RS4 but not RS2 induced phylum-level changes, significantly increasing Actinobacteria and Bacteroidetes while decreasing Firmicutes. At the species level, the changes evoked by RS4 were increases in Bifidobacterium adolescentis and Parabacteroides distasonis, while RS2 significantly raised the proportions of Ruminococcus bromii and Eubacterium rectale when compared to RS4. The population shifts caused by RS4 were numerically substantial for several taxa, leading for example, to a ten-fold increase in bifidobacteria in three of the subjects, enriching them to 18-30% of the fecal microbial community. The responses to RS and their magnitudes varied between individuals, and they were reversible and tightly associated with the consumption of RS.


Our results demonstrate that RS2 and RS4 show functional differences in their effect on human fecal microbiota composition, indicating that the chemical structure of RS determines its accessibility by groups of colonic bacteria. The findings imply that specific bacterial populations could be selectively targeted by well-designed functional carbohydrates, but the inter-subject variations in the response to RS indicates that such strategies might benefit from more personalized approaches.

Ketones and NKCC1/KCC2  
The next part of this post does get complicated and so it will be of interest to a smaller number of readers; the question is whether ketones play a role in the (miss) expression of those two critical chloride transporters NKCC1 and KCC2. If ketones play a role, then they might have therapeutic potential in all those people with autism and Down Syndrome who respond to bumetanide.
Some researchers think ketones play such a role but the bumetanide for autism researchers disagree.
The ongoing disagreement in the research is about the role played by a lack of ketones in why in immature neurons GABA is excitatory. Regular readers will know that in a large group of autism the GABA switch never flips and so neurons remain in the immature state that was only supposed to last weeks after birth. The debate in the research is to what extent ketone bodies play a role.
We know that in much autism and indeed in those with other problems like neuropathic pain, there can be elevated chloride due to over-expression of NKCC1 (through which chloride enters) and under-expression of KCC2 (through which chloride ions exit neurons). 

GABA action in immature neocortical neurons directly depends on the availability of ketone bodies. 


In the early postnatal period, energy metabolism in the suckling rodent brain relies to a large extent on metabolic pathways alternate to glucose such as the utilization of ketone bodies (KBs). However, how KBs affect neuronal excitability is not known. Using recordings of single NMDA and GABA-activated channels in neocortical pyramidal cells we studied the effects of KBs on the resting membrane potential (E(m)) and reversal potential of GABA-induced anionic currents (E(GABA)), respectively. We show that during postnatal development (P3-P19) if neocortical brain slices are adequately supplied with KBs, E(m) and E(GABA) are both maintained at negative levels of about -83 and -80 mV, respectively. Conversely, a KB deficiency causes a significant depolarization of both E(m) (>5 mV) and E(GABA) (>15 mV). The KB-mediated shift in E(GABA) is largely determined by the interaction of the NKCC1 cotransporter and Cl(-)/HCO3 transporter(s). Therefore, by inducing a hyperpolarizing shift in E(m) and modulating GABA signaling mode, KBs can efficiently control the excitability of neonatal cortical neurons.

In the early postnatal period, energy metabolism in the suckling rodent brain relies to a large extent on metabolic pathways alternate to glucose such as the utilization of ketone bodies (KBs). However, how KBs affect neuronal excitability is not known. Using recordings of single NMDA and GABA activated channels in neocortical pyramidal cells we studied the effects of KBs on the resting membrane potential (Em) and reversal potential of GABA-induced anionic currents (EGABA), respectively. We show that during postnatal development (P3–P19) if neocortical brain slices are adequately supplied with KBs, Em and EGABA are both maintained at negative

levels of about )83 and )80 mV, respectively. Conversely, a KB deficiency causes a significant depolarization of both Em (>5 mV) and EGABA (>15 mV). The KB-mediated shift in EGABA is largely determined by the interaction of the NKCC1 cotransporter and Cl)/HCO3 transporter(s). Therefore, by inducing a hyperpolarizing shift in Em and modulating GABA signaling mode, KBs can efficiently control the excitability of neonatal cortical neurons. Keywords: cortex, development, energy substrates, GABA, ketone bodies, resting potential.

showed that in the presence of KBs, values of EGABA in neocortical pyramidal neurons were close to Em, and did not change significantly during postnatal development, being maintained at about )80 mV (see Fig. 3). We cannot exclude the possibility that these values may differ in dendritic (Gulledge and Stuart 2003) or axonal (Price and Trussell 2006; Trigo et al. 2007; Khirug et al. 2008) compartments, an issue for future studies. Additionally, in this study we have limited our investigations to pyramidal cells, and the effects of KBs on interneurons remain to be explored. Nevertheless, the present observations suggest that energy substrates in the developing brain are an important issue to consider when studying neonatal neuronal excitability. Indeed, the most straightforward explanation for the difference between the results of the current study and those of previous studies of the development of neonatal GABA signaling lies in the fact that the brain of the suckling rodent relies strongly on KBs (Cremer and Heath 1974; Dombrowski et al. 1989; Hawkins et al. 1971; Lockwood and Bailey 1971; Lust et al. 2003; Page et al. 1971; Pereira de Vasconcelos and Nehlig 1987; Schroeder et al. 1991; Yeh and Zee 1976). Glucose utilization is limited at this age (Dombrowski et al. 1989; Nehlig, 1997; Nehlig et al. 1988; Prins 2008) because of the delayed maturation of the glycolytic enzymatic system (Dombrowski et al. 1989; Land et al. 1977; Leong and Clark 1984; Prins 2008). Use of glucose as the sole energy substrate caused an increase in neonatal neuronal [Cl)]i in our experiments, similar to that observed previously, while the addition of KBs resulted in a hyperpolarizing shift in both Em and EGABA. These results highlighted the need for caution in the interpretation of results obtained from neonatal brain slices superfused with standard ACSF. The cation chloride cotransporters NKCC1 and KCC2have been suggested to be the main regulators of neuronal Cl) homeostasis both during development (Farrant and Kaila 2007; Fiumelli and Woodin 2007) and in pathology (Galanopoulou, 2007; Kahle and Staley, 2008; Kahle et al. 2008).Although the possible contribution of anion exchangers to neuronal Cl) homeostasis has been noted previously (Farrant and Kaila 2007; Hentschke et al. 2006; Hubneret al. 2004; Pfeffer et al. 2009), they have not attracted the same degree of attention. Results from our study demonstrate, however, that the Cl)/HCO)3 transporter system is strongly involved in the KB-mediated regulation of [Cl)]i during postnatal development. Within this family, the Na-dependent Cl)/HCO)3 transporter (NDCBE), is of particular interest as it is expressed in the cortex (Chen et al. 2008) and has a strong dependence on ATP for its action (Chen et al. 2008; Davis et al. 2008; Romero et al. 2004). In addition, the sodium driven chloride bicarbonate exchanger(NCBE),(Giffardet al. 2003; Hubner et al. 2004; Lee et al. 2006) was expressed in the brain early during prenatal development and its expression preceded that of KCC2 (Hubner et al. 2004).

In neonatal neocortical neurons the interaction of NKCC1 and the Cl)/HCO3) transporter(s) maintained [Cl)]i, with KCC2 playing a less significant role at this stage. In the absence of KBs, when Cl)/HCO3) transporter(s) were less effective, the role of NKCC1 as a Cl) loader was especially noticeable and resulted in a depolarizing EGABA. During development the contribution of KCC2 to neocortical neuronal Cl) homeostasis is likely to increase (Stein et al. 2004; Zhang et al. 2006), and the balance between the actions of the different Cl) transporters in adults should be studied in the future. In humans, blood levels of KBs increase considerably during fasting, strenuous exercise, stress, or on the high-fat, low-carbohydrate ketogenic diet (KD) (Newburgh and Marsh 1920). A rapidly growing body of evidence indicates that the KD can have numerous neuroprotective effects (Gasior et al. 2006). During treatment with the KD, levels of KBs increase in both blood and brain, and cerebral metabolism adapts to preferentially use KBs as an alternate energy substrate to glucose (Kim do and Rho 2008). In children, the KD has been used as an effective treatment for medically refractory epilepsy (Freeman et al. 2007; Hartman and Vining 2007). However, despite nearly a century of use, the mechanisms underlying its clinical efficacy have proved elusive (Morris 2005; Bough and Rho 2007; Kim do and Rho 2008). Suckling rodents provide a natural model of the KD because of the high ketogenic ratio (Wilder and Winter 1922) of rodent milk (Page et al. 1971; Nehlig 1999). We propose that the KB-induced modulation of GABA-signaling may constitute a mechanism of anticonvulsive actions of the KD.

Now for the opposing views: -

Brain slices incubated with glucose have provided most of our knowledge on cellular, synaptic, and network driven mechanisms. It has been recently suggested that γ‐aminobutyric acid (GABA) excites neonatal neurons in conventional glucose‐perfused slices but not when ketone bodies metabolites, pyruvate, and/or lactate are added, suggesting that the excitatory actions of GABA are due to energy deprivation when glucose is the sole energy source. In this article, we review the vast number of studies that show that slices are not energy deprived in glucose‐containing medium, and that addition of other energy substrates at physiologic concentrations does not alter the excitatory actions of GABA on neonatal neurons. In contrast, lactate, like other weak acids, can produce an intracellular acidification that will cause a reduction of intracellular chloride and a shift of GABA actions. The effects of high concentrations of lactate, and particularly of pyruvate (4–5 mm), as used are relevant primarily to pathologic conditions; these concentrations not being found in the brain in normal “control” conditions. Slices in glucose‐containing medium may not be ideal, but additional energy substrates neither correspond to physiologic conditions nor alter GABA actions. In keeping with extensive observations in a wide range of animal species and brain structures, GABA depolarizes immature neurons and the reduction of the intracellular concentration of chloride ([Cl]i) is a basic property of brain maturation that has been preserved throughout evolution. In addition, this developmental sequence has important clinical implications, notably concerning the higher incidence of seizures early in life and their long‐lasting deleterious sequels. Immature neurons have difficulties exporting chloride that accumulates during seizures, leading to permanent increase of [Cl]i that converts the inhibitory actions of GABA to excitatory and hampers the efficacy of GABA‐acting antiepileptic drugs.  

GABA depolarizes immature neurons because of a high [Cl]i and orchestrates giant depolarizing potential (GDP) generation. Zilberter and coworkers (Rheims et al., 2009; Holmgren et al., 2010) showed recently that the ketone body metabolite dl-3-hydroxybutyrate (dl-BHB) (4 mm), lactate (4 mm), or pyruvate (5 mm) shifted GABA actions to hyperpolarizing, suggesting that the depolarizing effects of GABA are attributable to inadequate energy supply when glucose is the sole energy source. We now report that, in rat pups (postnatal days 4–7), plasma d-BHB, lactate, and pyruvate levels are 0.9, 1.5, and 0.12 mm, respectively. Then, we show that dl-BHB (4 mm) and pyruvate (200 μm) do not affect (i) the driving force for GABAA receptor-mediated currents (DFGABA) in cell-attached single-channel recordings, (2) the resting membrane potential and reversal potential of synaptic GABAA receptor-mediated responses in perforated patch recordings, (3) the action potentials triggered by focal GABA applications, or (4) the GDPs determined with electrophysiological recordings and dynamic two-photon calcium imaging. Only very high non physiological concentrations of pyruvate (5 mm) reduced DFGABA and blocked GDPs. Therefore, dl-BHB does not alter GABA signals even at the high concentrations used by Zilberter and colleagues, whereas pyruvate requires exceedingly high non-physiological concentrations to exert an effect. There is no need to alter conventional glucose enriched artificial CSF to investigate GABA signals in the developing brain.

Very recent research shows that ketones do not affect the expression of NKCC1 or KCC2. This would tend to support the argument of Ben Ari and Tyzio.

Nonetheless it seems that ketone bodies do indeed have an effect on GABA; they appear to change the hippocampal GABA/Glutamate ratio. 
So Tyzio might not be as right as he thought in his rebuttal paper when he said.
“suggesting, contrary to Zilberter and colleagues, that the antiepileptic actions of ketone bodies are not mediated by GABA signalling
Tyzio is thinking about the resting membrane potential (Em) and reversal potential of GABA-induced anionic currents (EGABA).
At the end of that day a reduction in gamma-glutamylated amino acids, caused by changes in gut microbiota cause an increase in hippocampal GABA/Glutamate ratio. If you happen to have epilepsy this may mean less seizures.  

I think we can say with a fair degree of certainty that we now know why the ketogenic diet, and indeed the modified Atkins diet, greatly reduce seizures in many people with epilepsy. The diet changes the gut microbiota by increasing the amount of Akkermansia muciniphila and Parabacteroides species, the end result is an increase in GABA, the inhibitory neurotransmitter, inside the brain. In much epilepsy, more inhibition to neurons firing results is far less seizures.
GABA plays a key role in autism, albeit a complex one.
The ketone driven changes to GABA might explain why some people with autism respond to the KD or just ketone supplements, but ketones have many other effects relevant to autism that will be reviewed in later posts.

Friday, 29 June 2018

Oxaloacetate and Pepping up Bioenergetic Fluxes in Autism and other Neurological Diseases

BHI as a dynamic measure of the response of the body to stress
In this scheme, healthy subjects have a high BHI with a high bioenergetic reserve capacity, high ATP-linked respiration (AL) and low proton leak (PL). The population of mitochondria is maintained by regenerative biogenesis. During normal metabolism, a sub-healthy mitochondrial population, still capable of meeting the energetic demand of the cell, accumulates functional defects, which can be repaired or turned over by mitophagy. Chronic metabolic stress induces damage in the mitochondrial respiratory machinery by progressively decreasing mitochondrial function and this manifests as low ATP-linked respiration, low reserve capacity and high non-mitochondrial (e.g. ROS generation) respiration. These bioenergetically inefficient damaged mitochondria exhibit increased proton leak and require higher levels of ATP for maintaining organelle integrity, which increases the basal oxygen consumption. In addition, chronic metabolic stress also promotes mitochondrial superoxide generation leading to increased oxidative stress, which can amplify mitochondrial damage, the population of unhealthy mitochondria and basal cellular energy requirements. The persistence of unhealthy mitochondria damages the mtDNA, which impairs the integrity of the biogenesis programme, leading to a progressive deterioration in bioenergetic function, which we propose can be identified by changes in different parameters of the bioenergetics profile and decreasing BHI.

Source:  The Bioenergetic Health Index: a new concept in mitochondrial translational research

Bioenergetic is today’s new buzz word; it is again all about getting maximum power output (ATP) from your mitochondria, which we looked at from a different perspective in a recent post.

A simple lack of ATP inside the brain seems to be a feature of many kinds of neurological problem. 
Oxaloacetate (OA or OAA) is another interesting potential treatment for a wide range of neurological disorders from Alzheimer’s and ALS to Huntington’s and Parkinson’s. There are no effective treatments for any of these conditions and little has changed in decades.
OAA, at high doses, and in animal studies, does have some very interesting effects, but they are perhaps too wide ranging, because some may be helpful and others not. OAA is interesting but no panacea.
OAA is sold as a supplement in low doses. It changes so many things, I think it is not surprising that some people find it beneficial, whether it is for Bipolar, ADHD or something else.
I think at higher doses, where there is a measurable impact above and beyond the OAA you have naturally in your blood, there might be some benefit as a treatment for mitochondrial disease. That would mean most regressive autism and some Childhood Disintegrative Disorder (CDD).
So we can consider OAA as another potential therapy for bioenergetic dysfunction. We have come across many potential therapies already in this blog.
Here is a schematic summary of what OAA does.

OAA effects and inter-effect relationships. OAA, a bioenergetic intermediate, affects bioenergetic flux. This produces a number of molecular changes. CREB phosphorylation and CREB activity increase, which in turn promotes the expression of PGC1 family member genes. AMPK and p38 MAP phosphorylation increase, and these activated kinases enhance PGC1α co-activator function. PGC1-induced co-activation of the NRF1 transcription factor stimulates COX4I1 production, while PGC1-induced co-activation of the ERRα transcription factor stimulates VEGF gene expression (61). OAA-induced flux changes also stimulate the pro-growth insulin signaling pathway and reduce inflammation. The pro-growth effects of increased insulin pathway signaling and increased VEGF, in conjunction with a more favorable bioenergetic status and less inflammation, cooperatively stimulate hippocampal neurogenesis.

You may recall from earlier posts that PGC-1α is the master regulator of mitochondrial biogenesis. 
The PGC-1α protein also appears to play a role in controlling blood pressure, regulating cellular cholesterol homoeostasis, and the development of obesity.
The PGC-1 α protein interacts with the nuclear receptor PPARγ. PPARγ has been covered extensively in this blog; agonists of PPARγ do seem to be therapeutic in some autism. Many drugs that are used to treat Type 2 diabetes work because they are PPARγ agonists.
It is not a surprise that Oxaloacetate (OAA) lowers your blood sugar. 

Bioenergetics and bioenergetic-related functions are altered in Alzheimer's disease (AD) subjects. These alterations represent therapeutic targets and provide an underlying rationale for modifying brain bioenergetics in AD-affected persons. Preclinical studies in cultured cells and mice found that administering oxaloacetate (OAA), a Krebs cycle and gluconeogenesis intermediate, enhanced bioenergetic fluxes and upregulated some brain bioenergetic infrastructure-related parameters. We therefore conducted a study to provide initial data on the tolerability and pharmacokinetics of OAA in AD subjects. Six AD subjects received OAA 100 mg capsules twice a day for one month. The intervention was well-tolerated. Blood level measurements following ingestion of a 100 mg OAA capsule showed modest increases in OAA concentrations, but pharmacokinetic analyses were complicated by relatively high amounts of endogenous OAA. We conclude that OAA 100 mg capsules twice per day for one month are safe in AD subjects but do not result in a consistent and clear increase in the OAA blood level, thus necessitating future clinical studies to evaluate higher doses.

In addition to being proposed for the treatment of AD and diabetes, recent preclinical research has also identified OAA as a potential therapeutic agent for stroke, traumatic brain injury, amyotrophic lateral sclerosis, and glioma [15], [16], [17], [18]. The clinical safety data we now report should prove relevant to efforts intending to translate results from these preclinical studies to the clinical arena. Our study also informs our attempts to develop OAA as a treatment for AD. Overall, we conclude that although OAA 100 mg capsules twice per day for one month are safe in AD subjects, because a consistent and clear increase in the OAA plasma level was not observed future clinical studies need to evaluate higher doses.

Experimental: Oxaloacetate (OAA) active capsule containing 100 mg OAA and 100 mg ascorbate, taken daily  

Experimental: Part 2 - Oxaloacetate (OAA)2 gram/day 
Participants take 2 grams of OAA per day for period of 4 weeks

Participants take 2 capsules Jubilance 100 mg Oxaloacetate/150 mg Ascorbic Acid blend per day

Oxaloacetate is an energy metabolite found in every cell of the human body. It holds a key place in the Krebs Cycle within the mitochondria, providing energy to the cells. It is also a critical early metabolite in gluconeogenesis, which provides glucose for the heart and brain during times of low glucose. It is critical to human metabolism, proper cellular function and it is central to energy production and use in the body.
Oxaloacetate may affect Emotional PMS through multiple mechanisms. During PMS, there is a large increase in glucose utilization in the cerebellum of the brain in women who are affected with emotional mood swings. Oxaloacetate supplementation has been shown to support proper glucose levels in the body. Having an excess of oxaloacetate allows gluconeogenesis take place upon demand, thereby fueling the brain, and perhaps meeting cerebellum glucose need.
In addition to oxaloacetate's ability to support proper glucose regulation, oxaloacetate affects two chemicals in the brain, GABA and glutamate. Altering the GABA/Glutamate ratio can affect mood. Oxaloacetate supplementation can reduce glutamate levels in the brain via a process called "Glutamate Scavenging". In addition, oxaloacetate supplementation was shown to increase GABA levels in animal models. By both lowering glutamate and increasing GABA, the GABA/Glutamate ratio is affected, which may also help women with Emotional PMS.
This study will investigate oxaloacetate's effect on Emotional PMS using patient completed surveys to measure depression, anxiety, perceived stress, and aggression, and statistically compare these results against placebo (rice flour) and baseline measurements.

An interesting old paper from 1968 was recently highlighted to me by a friend, it  shows that sodium oxaloacetate is particularly effective in treating type-1 diabetes.  In type-2 diabetes the effect range from none/minor in most  to a profound effect in a minority.
The meaning of “treating” was reducing blood sugar levels.
This study was the result of identifying the active substance in the plant euonymus alatus sieb, which has known blood sugar lowering effects.

Introduced from northeast Asia in the 1860s. Widely planted as an ornamental and for highway beautification due to its reliable and very showy fall foliage coloration. Numerous cultivars are available.
Other states where invasive: CT, DE, IN, KY, MA, MD, MO, NH, NJ, OH, PA, RI, VA, WI, WV. Federal or state listed as noxious weed, prohibited, invasive or banned: CT, MA.

Here is the interesting Japanese paper from 1968: 

There are more recent studies on the Euonymus alatus plant:

Euonymus alatus (E. alatus) is a medicinal plant used in some Asian countries for treating various conditions including cancer, hyperglycemia, and diabetic complications. This review outlines the phytochemistry and bioactivities of E. alatus related to antidiabetic actions. More than 100 chemical constituents have been isolated and identified from E. alatus, including flavonoids, terpenoids, steroids, lignans, cardenolides, phenolic acids, and alkaloids. Studies in vitro and in vivo have demonstrated the hypoglycemic activity of E. alatus extracts and its certain constituents. The hypoglycemic activity of E. alatus may be related to regulation of insulin signaling and insulin sensitivity, involving PPARγ and aldose reductase pathways. Further studies on E. alatus and its bioactive compounds may help to develop new agents for treating diabetes and diabetic complications.

A total of 26 flavonoids have been isolated and identified from E. alatus. The main structure types include flavonoid, flavanone, and flavonol. The aglycones of flavonoid glycosides isolated from E. alatus include quercetin, kaempferol, naringenin, aromadendrene, and dihydroquercetin. The flavonoids are mainly distributed in the leaves and wings of E. alatus
There is no mention of oxaloacetic acid.
The active components in protecting experimental diabetic nephropathy as mentioned above have also been suggested to be concentrated in ethyl acetate and n-butanol fractions [36, 40], though the nature of these compounds is still not identified. 
Euonymus alatus (E. alatus) has been used as a folk medicine for diabetes in China for more than one thousand years. In order to identify major active components, effects of different fractions of E. alatus on the plasma glucose levels were investigated in normal mice and alloxan-induced diabetic mice. Our results show that ethyl acetate fraction (EtOAc Fr.) displayed significant effects on reducing plasma glucose. In oral glucose tolerance, EtOAc Fr. at 17.2 mg/kg could significantly decrease the blood glucose of both normal mice and diabetic mice. After 4 weeks administration of the EtOAc Fr, when compared with the diabetic control, there were significant difference in biochemical parameters, such as glycosylated serum protein, superoxide dismutase and malondial dehyde, triglyceride, and total cholesterol, between alloxan-induced diabetic mice and the control group. Additional histopathological studies of pancreatic islets also showed EtOAc Fr. has beneficial effects on diabetic mice. Chemical analysis with three-dimensional HPLC demonstrated that the major components from EtOAc Fr were flavonoids and phenolic acids, which had anti-oxidative effects on scavenging DPPH-radical in vitro. All these experimental results suggest that EtOAc Fr. is an active fraction of E. alatus and can prevent the progress of diabetes. The mechanism of E. alatus for glucose control may be by stimulating insulin release, improving glucose uptake and improving oxidative-stress.

Oxaloacetic acid
You already have Oxaloacetic acid in your body, you make it.
Oxaloacetic acid (also known as oxalacetic acid) is a metabolic intermediate in many processes that occur in animals. It takes part in the gluconeogenesis, urea cycle, glyoxylate cycle, amino acid synthesis, fatty acid synthesis and citric acid cycle. Oxaloacetate is also a potent inhibitor of Complex II.

This post was prompted by our reader LatteGirl, who was asking about the supplement BenaGene and ketones. BenaGene contains 100mg of OAA and the company behind it is the sponsor of some the current OAA clinical trials.
The BenaGene supplement is sold by some companies that sell ketone products, but I do not really see big connection between OAA and ketones.   
If you can materially increase the plasma level of OAA, you really would expect numerous changes to occur.
Depending on what might be wrong with you, OAA might provide a net benefit, but it all looks very hit and miss. 
Treatment of all neurological disorders from ALS, Alzheimer’s to depression currently is remarkably hit and miss. Most serious disorders have only very partially effective treatments, but they do get FDA approval nonetheless.
The OAA research suggests its effect is from “altered bioenergetic fluxes”. You might be wondering what this actually means, since it sounds like pseudoscientific mumbo jumbo. What this really means is that for one reason or another there is a shortfall in energy (ATP) to power your cells.

“Perturbed bioenergetic function, and especially mitochondrial dysfunction, is observed in brains and peripheral tissues of subjects with Alzheimer's disease (AD) and mild cognitive impairment (MCI) (1,2), a clinical syndrome that frequently represents a transition between normal cognition and AD dementia (3). Neurons are vulnerable to mitochondrial dysfunction due to their high energy demands and dependence on respiration to generate ATP (4). Mitochondrial dysfunction may, therefore, drive or mediate various AD pathologies.”

Impaired energy (ATP) production can be caused by a deficiency in one of the mitochondrial enzyme complexes (often complex 1), but it could be caused by too few mitochondria (each cell needs many) or it could be caused by a lack of fuel (glucose or ketones), or oxygen.
Glucose crosses the blood brain barrier via a transporter called GLUT1.
GLUT1 deficiency leads to epilepsy, cognitive impairment and a small head (microcephaly). It can be treated by adopting the ketogenic diet, where ketones replace glucose as the fuel for your brain and body.
Oxygen freely crosses the blood brain barrier, but sometimes there is not enough of it. To increase the amount of oxygen that is carried in the blood, mountaineers and the military sometimes use the drug Diamox, which changes the pH of your blood, among other effects.
The brain's blood supply is via microvasculature/microvessels. This does seem to be impaired in autism, according to the research, resulting in unstable blood flow to the brain. 
Thyroid hormones are generally seen as regulating your basal/resting metabolic rate, so rather like your idle on your car, when you do not press the accelerator/gas pedal.  If the idle rate is too low your car will stall in traffic.
Thyroid hormone has many other effects and these are very important in the brain, particularly during development. A lack of the T3 hormone will lead to a physically different brain, whereas in adulthood it just causes impaired function which is reversible.
Thyroid hormones T3 and T4 can cross the blood brain barrier. The prohormone T4 is converted into the active hormone T3 within the brain. Some research suggests that T4 may have a direct role in the brain, rather than simply being a source of T3.

Thyroid receptors in the brain
TRα1 encompasses 70–80% of all TR expression in the adult vertebrate brain and TRα1 is present in nearly all neurons
It appears that windows in brain development may exist where T4 itself may act on TRα1.
Thyroid Hormone (TH) endocrinology in the CNS is tightly regulated at multiple tiers. Negative feedback loops in the hypothalamus and the pituitary control T3 and T4 output by the thyroid gland itself. Further, multiple phenomenon functions together to modulate the transport of circulating TH through the BBB, and multiple transporters act together to directly alter TH availability in the CNS itself. Additionally, conversion of intracellular T4 into T3 by deiodinase 2, inactivation of both T3 and T4 by deiodinase 3, and, the ability of different TR isoforms and different coregulators to respond directly to T4 versus T3 further regulate the CNS response to TH. 

The thyroid hormone receptor subtypes TRα and TRβ are expressed throughout the brain from early development, and mediate overlapping actions on gene expression. However there are also TR-subtype specific actions. Dio3 for example is induced by T3 specifically through TRα1. In vivo T3 regulates gene expression during development from fetal stages, and in adult animals. A large number of genes are under direct and indirect regulation by thyroid hormone. In neural cells T3 may control around 5% of all expressed genes, and as much as one third of them may be regulated directly at the transcriptional level. Thyroid hormone deficiency during fetal and postnatal development may cause retarded brain maturation, intellectual deficits and in some cases neurological impairment. Thyroid hormone deficiency to the brain during development is caused by iodine deficiency, congenital hypothyroidism, and maternal hypothyroidism and hypothyroxinemia. The syndromes of Resistance to Thyroid Hormones due to receptor mutations, especially TRα, cause variable affectation of brain function. Mutations in the monocarboxylate transporter 8 cause a severe retardation of development and neurological impairment, likely due to deficient T4 and T3 transport to the brain.   

Thyroid hormones are essential for brain maturation, and for brain function throughout life. In adults, thyroid diseases can lead to various clinical manifestations (1,2). For example, hypothyroidism causes lethargy, hyporeflexia and poor motor coordination. Subclinical hypothyroidism is often associated with memory impairment. Hypothyroidism is also associated to bipolar affective disorders, depression, or loss of cognitive functions, especially in the elderly (3). Hyperthyroidism causes anxiety, irritability, and hyperreflexia. Both, hypothyroidism or hyperthyroidism can lead to mood disorders, dementia, confusion, and personality changes. Most of these disorders are usually reversible with proper treatment, indicating that thyroid hormone alterations of adult onset do not leave permanent structural defects.
The actions of thyroid hormone during development are different, in the sense that they are required to perform certain actions during specific time windows. Thyroid hormone deficiency, even of short duration may lead to irreversible brain damage, the consequences of which depend not only on the severity, but also on the specific timing of onset and duration of the deficiency (4-8).
Hypothyroidism causes delayed and poor deposition of myelin

Pep up your Bioenergetic Fluxes
Within this blog we have encountered a wide range of methods that might help put correct a deficiency in power available to the brain.
·      Improve brain microvasculature function (Agmatine)

·      Ensure central basal metabolic rate is high enough (T3 hormone)

·      Increase D2 (lower oxidative stress, kaempferol) if centrally hypothyroid

·      Increase number of mitochondria (activate PGC1alpha)

·      Ensure adequate mitochondrial enzyme complexes for OXPHOS

·      Ensure adequate glucose transport via GLUT1

While I still feel Bioenergetic Fluxes sounds like something very quack-like, it is the valid terminology and it does look important to many neurological conditions.
In Monty, aged 14 with ASD, Agmatine has worked wonders, in terms of being far more energetic. I assume the effect is via increasing eNOS (endothelial nitric oxide synthase) and this has improved blood supply. We saw that blood flow through microvasculature/microvessels is impaired in autism.  We also saw that in mouse model of Alzheimer’s, Agmatine has a similar positive effect; this also seems to apply in at least some humans with Alzheimer’s.  

We can certainly add Oxaloacetate to the long list of substances we have come across in this blog that may well be therapeutic in diabetes. In the case of Oxaloacetate, it is type-1 that seems to uniformly benefit, whereas in type-2 diabetes some benefit and some do not.
It is amazing that in type-1 diabetes, only insulin is routinely prescribed, when so many things can increase insulin sensitivity and reduce the severe complications of this type of diabetes.
In the case of type-2 diabetes, you can halt its progression and, for the really committed, we saw how you can reverse it.
If a common, life-threatening, condition like diabetes is not fully treated, no wonder nobody bothers to treat an amorphous condition like autism.