Showing posts with label Epilepsy. Show all posts
Showing posts with label Epilepsy. Show all posts

Tuesday, 30 July 2019

Ginseng Compound K Esters for some Epilepsy, Autism and Cancers?

Many natural products like Ginseng and Curcumin do have long known medicinal properties but suffer from extremely low bioavailability, which limits their benefit.
Ginsenosides are compounds found in the Ginseng plant. They are metabolized by the gut flora into active compounds that include Compound K.  Compound K has been shown to have a variety of pharmacological actions such as anti-inflammatory, anti-oxidant, anti-cancer and vasorelaxation.  It also has interesting effects that relate to autism and other neurological disorders.

Compound K (CK) has extremely low bioavailability (circa 5%) which limits it potential therapeutic benefit. There are expensive versions of ginseng that aim to maximize Compound K (CK) production in the gut, but they do nothing to improve how much gets from the gut into the bloodstream.

It is possible to modify Compound K by making an ester. This ester has been shown to be highly bioavailable and that means the theoretic benefits, shown in test tubes, might actually be genuinely achieved in humans.

Two types of ester have been studied, the butyl and octyl ester resulting in so-called CK-B and CK-O.

There currently is a $400 million business selling ginseng worldwide, the research and production is mainly coming from Korea and China.  There probably should be pharmaceutical production of CK-B and/or CK-O, but I would not hold your breath.

CK-O was recently proposed as a treatment for some cancers, so perhaps someone will commercialize it.

Interestingly, the standard form CK has been proposed to treat colon cancer, which does make sense since CK is produced in the gut making colon cancer a good choice. You would think that CK-O would work better.

Bcl-2 / Bax

The gene/protein Bcl-2 is relevant to both cancer and autism and has been covered previously in this blog.

A total of 25 genes make up the Bcl-2 family of proteins. Bcl-2 itself is anti-apoptotic while family member Bax is pro-apoptotic.

Apoptosis is programmed cell death.  The Bax/Bcl-2 ratio determines the apoptotic potential of a cell. Increasing the Bax/Bcl-2 ratio can be highly desirable if you have cancer, since what you want is cell death.

Bcl-2 is dysregulated in autism. Studies have shown that the expression of Bcl-2 is significantly decreased in the brain of autistic subjects. This means a reduction in a protein that blocks apoptotic cell death, i.e. this favours growth and too much growth is a bad thing.

The big head type of autism (macrocephaly) is associated with hyperactive pro-growth signalling pathways, so reduced expression of Bcl-2 is not a surprise.

CK-O for some cancer

The Compound K ester CK-O  exerts strong anti-tumour activity by suppression of anti-apoptotic protein Bcl-2 and increase of pro-apoptotic protein Bax. It increases the Bax/Bcl-2 ratio.

CK-O from some epilepsy and some autism?

There are many types of epilepsy and hundreds of types of autism.
One commonly shared feature is the imbalance between the GABA-mediated inhibition and the glutamate-mediated excitation.

CK-O looks likes it might help both conditions.

·        CK is shown to reduce the expression of NMDA receptors and to attenuate the function of the NMDA receptors in the brain.

·        GABAB receptor activation via CK can regulate KCC2 at the cell surface in a manner that reduces intracellular chloride and hence the reversal potential for GABAA receptors

·        The expression of KCC2 protein was elevated by the treatment of CK while the expression of NKCC1 protein was reversely down-regulated.

·        CK enhances the expression of GABAA receptor subunit α1 in the brain and exhibits a tendency to decrease the expression of NMDAR1 protein in the hippocampus.

Ginseng for Autism?

There is some weak evidence that Ginseng may help in some autism.

I think what is really happening is that the effect is weak rather than the evidence is weak.  Ginseng may have a weak positive effect in some autism; weak because the amount of Compound K absorbed is trivial.

If Ginseng helps, CK-O could be substantially more effective.





I would not expect a dramatic effect from any commercial Ginseng product, but CK-O really could have an effect.

Although there are many research reports regarding the bioactive function of ginsenosides and ginseng, studies on the neuroprotective eect and eects on the cognitive function of compound K are limited. It is generally agreed that compound K is more bioavailable than the parent ginsenosides, including Rb1, Rb2, and Rc, and is the major contributing factor to the health benefits of ginseng. However, as most studies were conducted using disease-associated models, such as Alzheimer’s disease and ischemic stroke, the results cannot be directly translated to the healthy normal population. Furthermore, it is not clear whether compound K can cross the blood–brain barrier and exert any action on cognitive function in humans, even though the compound was reported to facilitate GABA release in the hippocampus and exhibit a protective eﬀect against scopolamine-induced hippocampal damage in a mouse model. The possible mechanisms of action of compound K in neuroprotection and cognitive improvement include attenuation of ROS levels in neural cells through induction of antioxidant enzymes, regulation of NO, GABA, and serotonin receptors, Ca 2+ channel modulation, regulation of the MAPK pathway, and inhibition of inflammation.
Although ginseng and ginsenosides were shown to have neuroprotective and cognitive enhancing eects, further research is required to establish whether compound K is the major component of ginseng responsible for cognitive improvement in humans.

The imbalance between the GABA-mediated inhibition and the glutamate-mediated excitation is the primary pathological mechanism of epilepsy. GABAergic and glutamatergic neurotransmission have become the most important targets for controlling epilepsy. Ginsenoside compound K (GCK) is a main metabolic production of the ginsenoside Rb1, Rb2, and Rc in the intestinal microbiota. Previous studies show that GCK promoted the release of GABA from the hippocampal neurons and enhanced the activity of GABAA receptors. GCK is shown to reduce the expression of NMDAR and to attenuate the function of the NMDA receptors in the brain. The anti-seizure effects of GCK have not been reported so far. Therefore, this study aimed to investigate the effects of GCK on epilepsy and its potential mechanism. The rat model of seizure or status epilepticus (SE) was established with either Pentylenetetrazole or Lithium chloride-pilocarpine. The Racine’s scale was used to evaluate seizure activity. The levels of the amino acid neurotransmitters were detected in the pilocarpine-induced epileptic rats. The expression levels of GABAARα1, NMDAR1, KCC2, and NKCC1 protein in the hippocampus were determined via western blot or immunohistochemistry after SE. We found that GCK had deceased seizure intensity and prolonged the latency of seizures. GCK increased the contents of GABA, while the contents of glutamate remained unchanged. GCK enhanced the expression of GABAARα1 in the brain and exhibited a tendency to decrease the expression of NMDAR1 protein in the hippocampus. The expression of KCC2 protein was elevated by the treatment of GCK after SE, while the expression of NKCC1 protein was reversely down-regulated. These findings suggested that GCK exerted anti-epileptic effects by promoting the hippocampal GABA release and enhancing the GABAAR-mediated inhibitory synaptic transmission.

Absorption mechanismof ginsenoside compound K and its butyl and octyl ester prodrugs in Caco-2cells.


Ginsenoside compound K (CK) is a bioactive compound with poor oral bioavailability due to its high polarity, while its novel ester prodrugs, the butyl and octyl ester (CK-B and CK-O), are more lipophilic than the original drug and have an excellent bioavailability. The aim of this study was to examine the transport mechanisms of CK, CK-B, and CK-O using human Caco-2 cells. Results showed that CK had a low permeability coefficient (8.65 × 10(-7) cm/s) for apical-to-basolated (AP-BL) transport at 10-50 μM, while the transport rate for AP to BL flux of CK-B (2.97 × 10(-6) cm/s) and CK-O (2.84 × 10(-6) cm/s) was significantly greater than that of CK. Furthermore, the major transport mechanism of CK was found as passive transcellular diffusion with active efflux mediated by P-glycoprotein (P-gp). In addition, it was found that CK-B and CK-O were not the substrate of efflux transporter since the selective inhibitors (verapamil and MK-571) of efflux transporter had little effects on the transport of CK-B and CK-O in the Caco-2 cells. These results suggest that improving the lipophilicity of CK by acylation can significantly improve the transport across Caco-2 cells.

Panax ginseng C.A. Meyer, the active components of which are mainly ginsenosides, is frequently utilized as a herbal drug in traditional oriental medicine. These ginsenosides, which belong to the class of triterpene saponins, have been reported to possess various biological and pharmacological activities such as antiaging, antiinflammation and antioxidation in central nerve system, cardiovascular system and immune system. Previous studies have shown that the pharmacological actions of ginsenosides contributed to their metabolites through biotransformation by human intestinal bacteria. Compound K (CK; Figure 1) is one of the main pharmacologically active metabolites of protopanaxadiol ginsenosides (e.g., Rb1, Rb2 and Rc) and it was reported that, it was accumulated in the liver after absorption from the GI tract to the blood, and some CK was transformed into fatty acid esters which may be the active components of ginsenosides in the body. Many studies revealed that most of the ginsenosides are poorly absorbed along the human intestinal tract due to a high polarity. Odani et al. have reported that the amount of ginsenoside Rg1 absorbed via oral administration was within the range of 1.9−20.0% of the dosage in animal models. Other ginsenosides such as Rb1 and Rb2 were also slowly absorbed through digestive tract, and the oral bioavailabilities in rats were relatively low. The biological activities of drugs depend not only on their chemical structures, but also on their degree of lipophilic and membrane permeation, which could enhance their transport across the cell membrane or influence their interaction with proteins and enzymes. Recently, considerable attention has been paid to the development of ester prodrugs, which is a widely used approach to improving overall lipophicity, membrane permehave been reported to enhance its lipophilicity, bioavailability and in vivo activity. However, to date, limited information is available concerning the mechanisms of oral absorption for CK and production of ester prodrugs to enhance the oral absorption of ginsenoside CK. To increase the oral absorption of CK, esterification provides a route to obtain more lipophilic derivatives. In addition, it has been reported that acylation of cholestane glycoside increased the antitumor potency. Several acylated triterpenoid saponins isolated from the roots of Solidago virgaurea subsp. virgaurea in a low concentration also activated the metabolism of endothelial cells, which enhanced the permeability of the blood vessel walls for better adsorption of the saponins into tissues. We thus speculated that the novel ester prodrugs of CK, butyl and octyl esters (CK-B and CK-O; Figure 1), which are more lipophilic than parent compound, may have an excellent oral bioavailability. The objective of this study was to determine the transepithelial transport and absorption mechanisms of CK and its ester derivatives in the Caco-2 system. Caco-2 cell monolayers have been generally accepted as an in vitro model for prediction of drug absorption across human intestine and for mechanistic studies of intestinal drug transport since these cells show morphological and functional similarities to human small intestinal epithelial cells. In this study, both ester derivatives were utilized for transepithelial transport and absorption assays in Caco-2 monolayers compared with CK to investigate whether esterification could enhance the membrane permeability of high hydrophilic compound, thus improving the intestinal absorption of drug.
Our results are consistent with the previous reports which showed that CK had a low oral bioavailability (approximately 5%) in rats. However, as shown in our results, the low oral bioavailability of CK can be improved by esterification of CK into CK-B and CK-O.

Octyl ester ofginsenoside compound K as novel anti‐hepatoma compound: Synthesis and evaluation on murine H22 cells in vitro and in vivo

Ginsenoside compound K (M1) is the active form of major ginsenosides deglycosylated by intestinal bacteria after oral administration. However, M1 was reported to selectively accumulate in liver and transform to fatty acid esters. Ester of M1 was not excreted by bile as M1 was, which means it was accumulated in the liver longer than M1. This study reported a synthetic method of M1‐O, a mono‐octyl ester of M1, and evaluated the anticancer property against murine H22 cell both in vitro and in vivo. As a result, both M1 and M1‐O showed a dose‐dependent manner in cytotoxicity assay in vitro. At lower dose of 12.5 μm, M1‐O showed moderate detoxification. Instead, M1‐O exhibited significantly higher inhibition in H22‐bearing mice than M1. M1‐O induced murine H22 tumor cellular apoptosis in caspase‐dependent pathway given that pan‐caspase inhibitor, Z‐VAD‐FMK, could reverse the cytotoxicity induced by M1‐O. Additionally, pro‐ and anti‐apoptosis proteins, Bcl‐2 and Bax, altered and consequently induced increased expression of cleaved caspase‐3. Interestingly, cyclophosphamide regimen significantly induced atrophy of spleen and thymus, main immune organs, while M1‐O treatment greatly alleviated this atrophy. Collectively, we propose M1‐O as a candidate for live cancer treatment.

M1-O exerted strong anti-tumor activity by suppression of anti-apoptotic protein Bcl-2 and increase of pro-apoptotic protein Bax

Note: M1-O is the same think as CK-O

Ginsenosides are isolated from the Panax quinquefolius. This is a natural product triterpene saponins and steroid glycosides. Ginsenosides are the members of a dammarane family, which consists of a 4-ring and steroid-like structure. All ginsenosides have two or three hydroxyl groups in the carbon 3 and 20. Ginsenosides are converted into active metabolites like 20(S)- protopanaxadiol Rb1-Rb3, Rc, Rd, Rg3, Rh2, Rs1 (2) with help of human gut bacteria -glycosidase Eubacterium sp. A-44. Ginsenosides produced a variety of pharmacological activities such as anti-inflammatory, anti-oxidant, anti-cancer and vasorelaxation.                                                                                                                                                                       

Emerging signals modulating potential of ginseng and its active compounds focusing on neurodegenerative diseases



The ginseng compound K ester CK-O is likely to be a potent drug in humans with a range of effects, some of which do relate to autism and epilepsy.

Very often people with epilepsy are excluded from autism clinical trials.  Here is one drug where you might want to start with that very group.

CK-O will have multiple effects, meaning it is not selective, so while it may have some very good effects, there may be some negative ones.

You might think the CK-O molecule would be a good basis on which to build a modern patentable drug; a K-O (knock-out) for someone.

Natural substances with health benefits like phytoestrogens (soy etc), curcumin/turmeric, ginseng and even bee propolis either need to be eaten in large quantities or the active substance identified and synthesized. The people with neurofibromatosis (NF-1, NF-2) consuming large amounts of expensive New Zealand propolis as a PAK1 inhibitor might as well save money and buy the active ingredient itself another ester, this time caffeic acid phenethyl ester, and gives the bees a rest.

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).


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.




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.




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). 



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.