Making best use of existing NKCC1/2 Blockers in Autism

Azosemide C12H11ClN6O2S2  

Source: https://pubchem.ncbi.nlm.nih.gov/compound/azosemide

Today’s post may be of interest to those already using bumetanide for autism and for those considering doing so.  It does go into the details, because they really do matter and does assume some prior knowledge from earlier posts.

There has been a very thorough new paper published by a group at Johns Hopkins:-

Off-Label Use of Bumetanide for Brain Disorders: An Overview

It does cover all the usual issues and raises some points that have not been covered yet in this blog.  One point is treating autism prenatally. This issue was studied twice in rats, and the recent study was sent to me by Dr Ben Ari.  Short term treatment during pregnancy produced a permanent benefit.

Maternal bumetanide treatment prevents the overgrowth in the VPA condition

            

Brief maternal administration of bumetanide before birth restores low neuronal intracellular chloride concentration ([Cl⁻]_i) levels, produces an excitatory-to-inhibitory shift in the action of γ-aminobutyric acid (GABA), and attenuates the severity of electrical and behavioral features of ASD (9, 10), suggesting that [Cl⁻]_i levels during birth might play an important role in the pathogenesis of ASD (7). Here, the same bumetanide treatment significantly reduced the hippocampal and neocortical volumes of P0 VPA pups, abolishing the volume increase observed during birth in the VPA condition [hippocampus: P0 VPA versus P0 VPA + BUM (P = 0.0116); neocortex: P0 VPA versus P0 VPA + BUM (P = 0.0242); KWD] (Fig. 3B). Maternal bumetanide treatment also shifted the distribution of cerebral volumes from lognormal back to normal in the population of VPA brains, restoring smaller cerebral structure volumes (Fig. 3C). It also decreased the CA3 volume to CTL level after birth, suggesting that the increased growth observed in this region could be mediated by the excitatory actions of GABA (Fig. 3D). Therefore, maternal bumetanide administration prevents the enhanced growth observed in VPA animals during birth.

One issue with Bumetanide is that it affects both:-

·        NKCC2 in your kidneys, causing diuresis

·        NKCC1 in your brain and elsewhere, which is divided into two slightly different forms NKCC1a and NKCC1b

NKCC1 is also expressed in your inner ear where it is necessary for establishing the potassium-rich endolymph that bathes part of the cochlea, an organ necessary for hearing. 

If you block NKCC1 too much you will affect hearing.

Blocking NKCC1 in children and adults is seen as safe but the paper does query what the effect on hearing might be if given prenatally as the ear is developing.

Treating Down Syndrome Prenatally

While treating autism prenatally might seem a bit unlikely, treating Down Syndrome (DS) prenatally certainly is not.  Very often DS is accurately diagnosed before birth creating a valuable treatment window.  In most countries the vast majority of DS prenatal diagnoses lead to termination, but only a small percentage of pregnancies are tested for DS. In some countries such as Ireland a significant number of DS pregnancies are not terminated, these could be treated to reduce the deficits that will otherwise inevitably follow.

Timing of therapies for Down syndrome: the sooner, the better

The research does suggest that DS is another brain disorder that responds to bumetanide.

Back to autism and NKCC1

This should remind us that a defect in NKCC1 expression will not only cause elevated levels of chloride with in neurons, but will also affect the levels of sodium and potassium with neurons.

There are many ion channel dysfunctions (channelopathies) implicated in autism and elevated levels of sodium and potassium will affect numerous ion channels.  The paper does suggest that the benefit of bumetanide may go beyond modifying the effect of GABA, which is the beneficial mode of action put forward by Dr Ben Ari.

We have seen how hypokalemic sensory overload looks very similar to what often occurs in autism and that autistic sensory overload is reduced by taking an oral potassium supplement.

The paper also reminds us that loop diuretics like bumetanide and furosemide not only reduce inflow of chloride into neurons, but may also reduce the outflow. This is particularly known of furosemide, but also occurs with bumetanide at higher doses.

The chart below shows that the higher the concentration of bumetanide the strong its effect becomes on blocking NKCC1.

But at higher doses there will also be a counter effect of closing the NKCC2 transporter that allows chloride to leave neurons.

At some point a higher dose of bumetanide may have a detrimental effect on trying to lower chloride within neurons.

Since Dr Ben Ari’s objective is to lower chloride levels in neurons  it is important how freely these ions both enter and exit.  The net effect is what matters. (Loop diuretics block NKCC1 that lets chloride enter neurons but also block the KCC2 transporter via which they exit)

Is Bumetanide the optimal existing drug to lower chloride within neurons?  Everyone agrees that it is not, because only a tiny amount crosses into the brain. The paper gives details of the prodrugs like BUM5 that have been looked at previously in this blog; these are modified versions of bumetanide that can better slip across the blood brain barrier and then react in the brain to produce bumetanide itself.  It also highlights the recent research that suggests that Bumetanide may not be the most potent approved drug, it is quite conceivable that another old drug called Azosemide is superior.

The blood brain barrier is the problem, as is often the case.  Bumetanide has a low pH (it is acidic) which hinders its diffusion across the barrier.  Only about 1% passes through.

There is scepticism among researchers that enough bumetanide can cross into the brain to actually do any good.  This is reflected in the review paper.

The paper reminds us of the research showing how you can boost the level of bumetanide in the brain by adding Probenecid, an OAT3 inhibitor.  During World War 2 antibiotics were in short supply and so smaller doses were used, but their effect was boosted by adding Probenecid. By blocking OAT3, certain types of drug like penicillin and bumetanide are excreted at a slower rate and so the net level in blood increases.

The effect of adding Probenecid, or another less potent OAT3 inhibitors, is really no different to just increasing the dose of bumetanide.

The problem with increasing the dose of bumetanide is that via its effect on NKCC2 you cause even more diuresis, until eventually a plateau is reached.

Eventually, drugs selective for NKCC1a and/or NKCC1b will appear.

In the meantime, the prodrug BUM5 looks good. It crosses the BBB much better than bumetanide, but it still affects NKCC2 and so will cause diuresis.  But BUM5 should be better than Bumetanide + Probenecid, or a higher dose of Bumetanide.  BUM5 remains a custom-made research drug, never used in humans.

I must say that what again stands out to me is the old German drug, Azosemide.

In a study previously highlighted in this blog, we saw that Azosemide is 4 times more potent than Bumetanide at blocking NKCC1a and NKCC1b.

Azosemide is more potent than bumetanide and various other loop diuretics to inhibit the sodium-potassium-chloride-cotransporter human variants hNKCC1A and hNKCC1B

Azosemide is used in Japan, where recent research shows it is actually more effective than other diuretics

Azosemide, a Long-acting Loop Diuretic, is Superior to Furosemide in Prevention of Cardiovascular Death in Heart Failure Patients Without Beta-blockade 

As is often the case, Japanese medicine has taken a different course to Western medicine.

Years of safety information has already been accumulated on Azosemide.  It is not an untried research drug. It was brought to market in 1981 in Germany. It is available as Diart in Japan made by Sanwa Kagaku Kenkyusho and as a cheaper generic version by Choseido Pharmaceutical. In South Korea Azosemide is marketed as Uretin.

In any other sector other than medicine, somebody would have thought to check by now if Azosemide is better than Bumetanide.  It is not a matter of patents, Ben-Ari has patented all of the possible drugs, including Azosemide and of course Bumetanide.

So now we move on to Azosemide.

When researchers came to check the potency of the above drugs the results came as a surprise.  It turns out that the old German drug Azosemide is 4 times as potent as bumetanide.

The big question is how does it cross the blood brain barrier.

“The low brain concentrations of bumetanide obtained after systemic administration are thought to result from its high ionization (>99%) at physiological pH and its high plasma protein binding (>95%), which restrict brain entry by passive diffusion, as well as active efflux transport at the blood-brain barrier(BBB). The poor brain penetration of bumetanide is a likely explanation for its controversial efficacy in the treatment of brain diseases”

“… azosemide was more potent than any other diuretic, including bumetanide, to inhibit the two NKCC1 variants. The latter finding is particularly interesting because, in contrast to bumetanide, which is a relatively strong acid (pKa = 3.6), azosemide is not acidic (pKa = 7.38), which should favor its tissue distribution by passive diffusion. Lipophilicity (logP) of the two drugs is in the same range (2.38 for azosemide vs. 2.7 for bumetanide). Furthermore, azosemide has a longer duration of action than bumetanide, which results in superior clinical efficacy26 and may be an important advantage for treatment of brain diseases with abnormal cellular chloride homeostasis.”

Dosage equivalents of loop Diuretics

Bumetanide has very high oral bioavailablity, meaning almost all of what you swallow as a pill makes it into your bloodstream.

Furosemide and Azosemide have much lower bioavailability and so higher doses are needed to give the same effect.

Both Furosemide and Bumetanide are short acting, while Azosemide is long acting.

For a drug that needs to cross the blood brain barrier small differences might translate into profoundly different effects.

The limiting factor in all these drugs is their effect on NKCC2 that causes diuresis.

1mg of bumetanide is equivalent to 40mg of furosemide.

2mg of bumetanide is equivalent to 80mg of furosemide.

The standard dose for Azosemide in Japan, where people are smaller than in the West, is 30 mg or 60mg. 

Research suggests that the same concentration of Azosemide is 4x more potent than Bumetanide at blocking NKCC1 transporters, other factors that matter include:-

·        How much of the oral tablet ends up in the bloodstream.

·        How long does it stay in the blood stream

·        How much of the drug actually crosses the blood brain barrier

·        How does the drug bind to the NKCC1 transporters in neurons

·        How rapidly is the drug excreted from the brain

·        What effect is there on the KCC2 transporter that controls the exit of chloride ions from neurons.

All of this comes down to which is more effective in adults with autism 2mg of bumetanide or 60mg of Azosemide.

The side effects, which are mainly diuresis and loss of electrolytes will be similar, but Azosemide is a longer acting drug and so there will be differences. In fact Azosemide is claimed to be less troublesome than Bumetanide in lower potassium levels in your blood.

Conclusion  

The open question is whether generic Azosemide is “better” than generic Bumetanide for treating brain disorders in humans.

I did recently ask Dr Ben-Ari if he is aware of any data on this subject. There is none.

Many millions of dollars/euros are being spent getting Bumetanide approved for autism, so it would be a pity if Azosemide turns out to be better. (Dr Ben Ari’s company Neurochlore wants to develop a new molecule that will cross the blood brain barrier, block NKCC1 and not NKCC2 and so will not cause diuresis).

The hunch of the researchers from Hanover, Germany seems to be that the old German drug Azosemide will be better than Bumetanide.

I wonder if doctors at Johns Hopkins / Kennedy Krieger have started to prescribe bumetanide off-label to their patients with autism.  Their paper shows that they have a very comprehensive knowledge of the subject.

===========

I suggest readers consult the full version of the Johns Hopkins review paper on Bumetanide, it is peppered with links to all the relevant papers.

Off-Label Use of Bumetanide for Brain Disorders: An Overview

Bumetanide (BTN or BUM) is a FDA-approved potent loop diuretic (LD) that acts by antagonizing sodium-potassium-chloride (Na-K-Cl) cotransporters, NKCC1 (SLc12a2) and NKCC2. While NKCC1 is expressed both in the CNS and in systemic organs, NKCC2 is kidney-specific. The off-label use of BTN to modulate neuronal transmembrane Cl⁻gradients by blocking NKCC1 in the CNS has now been tested as an anti-seizure agent and as an intervention for neurological disorders in pre-clinical studies with varying results. BTN safety and efficacy for its off-label use has also been tested in several clinical trials for neonates, children, adolescents, and adults. It failed to meet efficacy criteria for hypoxic-ischemic encephalopathy (HIE) neonatal seizures. In contrast, positive outcomes in temporal lobe epilepsy (TLE), autism, and schizophrenia trials have been attributed to BTN in studies evaluating its off-label use. NKCC1 is an electroneutral neuronal Cl⁻importer and the dominance of NKCC1 function has been proposed as the common pathology for HIE seizures, TLE, autism, and schizophrenia. Therefore, the use of BTN to antagonize neuronal NKCC1 with the goal to lower internal Cl⁻ levels and promote GABAergic mediated hyperpolarization has been proposed. In this review, we summarize the data and results for pre-clinical and clinical studies that have tested off-label BTN interventions and report variable outcomes. We also compare the data underlying the developmental expression profile of NKCC1 and KCC2, highlight the limitations of BTN’s brain-availability and consider its actions on non-neuronal cells.

Btn Pro-Drugs and Analogs

To improve BTN accessibility to the brain, pro-drugs with lipophilic and uncharged esters, alcohol and amide analogs have been created. These pro-drugs convert to BTN after gaining access into the brain. There was a significantly higher concentration of ester prodrug, BUM5 (N,N – dimethylaminoethyl ester), in mouse brains compared to the parent BTN (10 mg/kg, IV of BTN and equimolar dose of 13 mg/kg, IV of BUM5) (Töllner et al., 2014). BUM5 stopped seizures in adult animal models where BTN failed to work (Töllner et al., 2014; Erker et al., 2016). BUM5 was also less diuretic and showed better brain access when compared to the other prodrugs, BUM1 (ester prodrug), BUM7 (alcohol prodrug) and BUM10 (amide prodrug). BUM5 was reported to be more effective than BTN in altering seizure thresholds in epileptic animals post-SE and post-kindling (Töllner et al., 2014). Furthermore, BUM5 (13 mg/kg, IV) was more efficacious than BTN (10 mg/kg, IV) in promoting the anti-seizure effects of PB, in a maximal electroshock seizure model (Erker et al., 2016). Compared to BUM5 which was an efficacious adjunct to PB in the above mentioned study, BTN was not efficacious when administered as an adjunct (Erker et al., 2016). In addition to seizure thresholds, further studies need to be conducted to assess effects of BUM5 on seizure burdens, ictal events, duration and latencies.

Recently, a benzylamine derivative, bumepamine, has been investigated in pre-clinical models. Since benzylamine derivatives lack the carboxylic group of BTN, it results in lower diuretic activity (Nielsen and Feit, 1978). This prompted Brandt et al. (2018) to explore the proposed lower diuretic activity, higher lipophilicity and lower ionization rate of bumepamine at physiological pH. Since it is known that rodents metabolize BTN quicker than humans, the study used higher doses of 10 mg/kg of bumepamine similar to their previous BTN studies (Olsen, 1977; Brandt et al., 2010; Töllner et al., 2014). Bumepamine, while only being nominally metabolized to BTN, was more effective than BTN to support anticonvulsant effects of PB in rodent models of epilepsy. This GABAergic response, however, was not due to antagonistic actions on NKCC1; suggesting bumepamine may have an off-target effect, which remains unknown. However, the anticonvulsive effects of bumepamine, in spite of its lack of action on NKCC1, are to be noted. Additionally, in another study by the same group, it was shown that azosemide was 4-times more potent an inhibitor of NKCC1 than BTN, opening additional avenues for better BBB penetration and NKCC1-antagonizing compounds for potential neurological drug discovery (Hampel et al., 2018).

Conclusion

The beneficial effects of BTN reported in cases of autism, schizophrenia and TLE, given its poor-brain bioavailability are intriguing. The mechanisms underlying the effects of BTN, as a neuromodulator for developmental and neuropsychiatric disorders could be multifactorial due to prominent NKCC1 function at neuronal and non-neuronal sites within the CNS. Investigation of the possible off-target and systemic effects of BTN may help further this understanding with the advent of a new generation of brain-accessible BTN analogs.

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

Source: The Gut Microbiota Mediates the Anti-Seizure Effects of the Ketogenic Diet

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).

Gut bacteria play critical role in anti-seizure effects of ketogenic diet, UCLA scientists report

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:

Bloom Science launches to treat epilepsy with ketogenic diet-associated gut microbes

“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 Gut Microbiota Mediates the Anti-Seizure Effects of the Ketogenic Diet

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.

http://www.ingredion.us/ingredients/na/versafibe-2470-dietary-fiber-22457b00.html

http://resistantstarchfoundation.com/resistant-starch-type-4/

Strategies to promote abundance of Akkermansia muciniphila, an emerging probiotics in the gut, evidence from dietary intervention studies

Viable A. muciniphila and fructooligosaccharides contently promote A. muciniphila. 

Metformin and vancomycin also significantly promote A. muciniphila.

https://biofoundations.org/increasing-and-maintaining-akkermansia-muciniphila-for-a-healthy-gut-microbiome/

“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 “ 

Two Healthy Diets Modulate Gut Microbial Community Improving Insulin Sensitivity in a Human Obese Population

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.

Beneficial modulation of the gut microbiota

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

Abstract

BACKGROUND:

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.

METHODS AND FINDINGS:

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.

CONCLUSION:

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. 

Abstract

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: -

Excitatory action of GABA on immature neurons is not due to absence of ketone bodies metabolites or other energy substrates

Summary

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.  

Depolarizing Actions of GABA in Immature Neurons Depend Neither on Ketone Bodies Nor on Pyruvate

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 GABA_A receptor-mediated currents (DF_GABA) in cell-attached single-channel recordings, (2) the resting membrane potential and reversal potential of synaptic GABA_A 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 DF_GABA 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.

Ketogenic diet does not change NKCC1 and KCC2 expression in rat hippocampus

  

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.  

Conclusion

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.