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Showing posts with label BHB. Show all posts
Showing posts with label BHB. Show all posts

Wednesday 19 September 2018

Ketones and Autism Part 5 - BHB, Histone Acetylation Modification, BDNF Expression, PKA, PKB/Akt, Microglial Ramification, Depression and Kabuki Syndrome















Child displaying elongated eyelids typical of Kabuki syndrome
Source: Given by Parents of children pictured with purpose of representing children with kabuki on Wikipedia. 

The syndrome is named after its resemblance to Japanese Kabuki makeup.

As we have discovered in this blog, autism is just a condition where certain genes are over-expressed and other genes are under-expressed. Put like that makes it sound quite simple.

Methylation of histones can either increase or decrease transcription of genes. The subject is highly complex, but we can keep things simple.

The child in the photo above has Kabuki syndrome and is likely to exhibit features of autism.  In most cases this is the result of a lack of expression of the KMT2D/MLL2 gene which encodes a protein called Histone-lysine N-methyltransferase.  Unfortunately, this is quite an important protein, because it promotes the “opening of chromatin”.  It adds a “trimethylation mark to H3K4”, just think of it as a pink post-it on your DNA. 
We get H3K4me3, which is an epigenetic marker (me3, because it is trimethylation). H3K4me3 promotes gene activation and it can cause a relative imbalance between open and closed chromatin states for critical genes. It has been suggested that it may be possible to restore this balance with drugs that promote open chromatin states, such as histone deacetylase inhibitors (HDACi).
What all this means is that people with Kabuki start with under-expression of just one gene, but this leads to the miss-expression of numerous other genes. Because science has figured out what the KMT2D/MLL2 gene does, we can find ways of treating this syndrome.

BHB as an HDAC inhibitor and a treatment for Kabuki syndrome

HDAC inhibitors (HDACi) are also suggested as therapies for other single gene syndromes. We saw in an earlier post that in Pitt Hopkins syndrome people lack Transcription Factor 4 (TCF4). Too little TC4 is not good, but too much TC4 is one feature of schizophrenia.
We saw in the research that we can increase expression of TCF4 using a class 1 HDAC inhibitor and we can also activate the Wnt pathway, which can also be achieved by inhibiting GSK3 (all themes covered in this blog).
So, Pitt Hopkins therapies include: -
·        Wnt activation (covered extensively in this blog) this includes statins and GSK3 inhibitors like Lithium

·        HDAC inhibitors like valproic acid, some cancer drugs, sodium butyrate and indeed the ketone BHB
This also means that people with schizophrenia, and likely too much TCF4, might benefit from the opposite gene expression modification, so a Wnt inhibitor, these include some cheap, safe, drugs used to treat children with parasites (Mebendazole/ Niclosamide etc) and of course GSK3 activators.
It is interesting that after 500 posts of this amateur blog you can start to fit the science together and identify rational therapies for complex disorders and  note that these therapies have much wider application, either to milder conditions or discovering avenues to treat the opposite genetic variation.  The underlying biological themes are all reoccurring in different types of autism/schizophrenia/ bipolar and you do wonder why more has not been done by professionals to apply this knowledge. 500 posts may sound a lot, but for autism researchers this is their paid, full-time job, not just a hobby pastime.

But then again, Simon Baron-Cohen, Head of Cambridge University's Autism Research Centre, recently published an article in which he wrote:

"We at the Autism Research Centre have no desire to cure, prevent or eradicate autism ... As scientists, our agenda is simply to understand the causes of autism." 

Whose team is he playing for?

My conclusion is that perhaps Baron-Cohen has Asperger's himself, because he does not realize that a disorder, severe enough for a medical/psychiatric diagnosis, is a bad thing that should be minimized and ideally prevented, just like any other brain disorder. His cousin the actor Sacha gives a very good impression of someone with bipolar, so perhaps they both need a Wnt activator?

Would a mother with Multiple Sclerosis (MS) want her daughter to also develop MS to share the experience? I think not. If it is just "quirky autism", it does not warrant a medical diagnosis, because it is perfectly okay to be quirky. 

This blog does have many Aspie readers who do want pharmacological therapy and that is their choice; I am fully supportive of them and wish them well.

Back to Kabuki
There is more than one type of HDAC and so there are different types of HDACi.  There are actually 18 HDAC enzymes divided into four classes
The ketone BHB inhibits HDAC class I enzymes called HDAC2 and HDAC3
The good news is that the ketogenic diet, which produces BHB, does indeed show merit as a therapy for Kabuki.

Kabuki syndrome is caused by haploinsufficiency for either of two genes that promote the opening of chromatin. If an imbalance between open and closed chromatin is central to the pathogenesis of Kabuki syndrome, agents that promote chromatin opening might have therapeutic potential. We have characterized a mouse model of Kabuki syndrome with a heterozygous deletion in the gene encoding the lysine-specific methyltransferase 2D (Kmt2d), leading to impairment of methyltransferase function. In vitro reporter alleles demonstrated a reduction in histone 4 acetylation and histone 3 lysine 4 trimethylation (H3K4me3) activity in mouse embryonic fibroblasts from Kmt2d+/βGeo mice. These activities were normalized in response to AR-42, a histone deacetylase inhibitor. In vivo, deficiency of H3K4me3 in the dentate gyrus granule cell layer of Kmt2d+/βGeo mice correlated with reduced neurogenesis and hippocampal memory defects. These abnormalities improved upon postnatal treatment with AR-42. Our work suggests that a reversible deficiency in postnatal neurogenesis underlies intellectual disability in Kabuki syndrome.

Intellectual disability is a common clinical entity with few therapeutic options. Kabuki syndrome is a genetically determined cause of intellectual disability resulting from mutations in either of two components of the histone machinery, both of which play a role in chromatin opening. Previously, in a mouse model, we showed that agents that favor chromatin opening, such as the histone deacetylase inhibitors (HDACis), can rescue aspects of the phenotype. Here we demonstrate rescue of hippocampal memory defects and deficiency of adult neurogenesis in a mouse model of Kabuki syndrome by imposing a ketogenic diet, a strategy that raises the level of the ketone beta-hydroxybutyrate, an endogenous HDACi. This work suggests that dietary manipulation may be a feasible treatment for Kabuki syndrome.
 Although BHB has previously been shown to have HDACi activity (7, 21), the potential for therapeutic application remains speculative. Here, we show that therapeutically relevant levels of BHB are achieved with a KD modeled on protocols that are used and sustainable in humans (22, 23). In addition, we demonstrate a therapeutic rescue of disease markers in a genetic disorder by taking advantage of the BHB elevation that accompanies the KD.
Our findings that exogenous BHB treatment lead to similar effects on neurogenesis as the KD support the hypothesis that BHB contributes significantly to the therapeutic effect. In our previous study (6), the HDACi AR-42 led to improved performance in the probe trial of the MWM for both Kmt2d+/βGeo and Kmt2d+/+ mice (genotype-independent improvement). In contrast, KD treatment only led to improvement in Kmt2d+/βGeo mice (genotype-dependent improvement). This discrepancy may relate to the fact that AR-42 acts as an HDACi but also affects the expression of histone demethylases (24), resulting in increased potency but less specificity. Alternatively, because the levels of BHB appear to be higher in Kmt2d+/βGeo mice on the KD, the physiological levels of BHB might be unable to reach levels in Kmt2d+/+ mice high enough to make drastic changes on chromatin.
In addition to the effects seen on hippocampal function and morphology, we also uncovered a metabolic phenotype in Kmt2d+/βGeo mice, which leads to both increased BHB/AcAc and lactate/pyruvate ratios during ketosis; an increased NADH/NAD+ ratio could explain both observations. This increased NADH/NAD+ ratio may relate to a previously described propensity of Kmt2d+/βGeo mice toward biochemical processes predicted to produce NADH, including beta-oxidation, and a resistance to high-fat-diet–induced obesity (27). If this exaggerated BHB elevation holds true in patients with KS, the KD may be a particularly effective treatment strategy for this patient population; however, this remains to be demonstrated. Alterations of the NADH/NAD+ ratio could also affect chromatin structure through the action of sirtuins, a class of HDACs that are known to be NAD+ dependent (28). Advocates of individualized medicine have predicted therapeutic benefit of targeted dietary interventions, although currently there are few robust examples (2931). This work serves as a proof-of-principle that dietary manipulation may be a feasible strategy for KS and suggests a possible mechanism of action of the previously observed therapeutic benefits of the KD for intractable seizure disorder (22, 23).                   
Kabuki syndrome (KS) (Kabuki make-up syndrome, Niikawa-Kuroki syndrome) is a rare genetic disorder first diagnosed in 1981. Kabuki make-up syndrome (KMS) is a multiple malformation/intellectual disability syndrome that was first described in Japan but is now reported in many other ethnic groups. KMS is characterized by multiple congenital abnormalities: craniofacial, skeletal, and dermatoglyphic abnormalities; intellectual disability; and short stature. Other findings may include: congenital heart defects, genitourinary anomalies, cleft lip and/or palate, gastrointestinal anomalies including anal atresia, ptosis and strabismus, and widely spaced teeth and hypodontia. The KS is associated with mutations in the MLL2 gene in some cases were also observed deletions of KDM6A. This study describes three children with autism spectrum disorders (ASDs) and KS and rehabilitative intervention that must be implemented.

So what?
Unless you know someone with Kabuki syndrome, you might be wondering what does this matter to autism.
What is shows is that BHB/KD is sufficiently potent to be a viable HDAC inhibitor. 
We know that some cancer drug HDAC inhibitors are effective in some mouse models of autism. But these drugs usually have side effects. 

HDAC Inhibitors for which Cancer/Autism? 

BHB is safe endogenous substance, so it is a “natural” HDACi. 

The effect of HDAC2 and HDAC3 on BDNF 
Brain derived neurotropic factor (BDNF) is like brain fertilizer. In some types of autism, you would like more BDNF.
When you exercise you produce BHB and that goes on to trigger the release of BDNF. This process also involves NF-kB activation

Exercise induces beneficial responses in the brain, which is accompanied by an increase in BDNF, a trophic factor associated with cognitive improvement and the alleviation of depression and anxiety. However, the exact mechanisms whereby physical exercise produces an induction in brain Bdnf gene expression are not well understood. While pharmacological doses of HDAC inhibitors exert positive effects on Bdnf gene transcription, the inhibitors represent small molecules that do not occur in vivo. Here, we report that an endogenous molecule released after exercise is capable of inducing key promoters of the Mus musculus Bdnf gene. The metabolite β-hydroxybutyrate, which increases after prolonged exercise, induces the activities of Bdnf promoters, particularly promoter I, which is activity-dependent. We have discovered that the action of β-hydroxybutyrate is specifically upon HDAC2 and HDAC3, which act upon selective Bdnf promoters. Moreover, the effects upon hippocampal Bdnf expression were observed after direct ventricular application of β-hydroxybutyrate. Electrophysiological measurements indicate that β-hydroxybutyrate causes an increase in neurotransmitter release, which is dependent upon the TrkB receptor. These results reveal an endogenous mechanism to explain how physical exercise leads to the induction of BDNF.

Results: ROS was significantly increased in neurons after 6 hours of ketone incubation. However, after 24 hours, neurons show improved efficiency in ATP productions, upregulated expressions of antioxidant enzyme SOD2, and enhanced resistance to excitotoxicity. These effects were significantly abolished in neurons after treatment with TrkB inhibitor. More interestingly, ROS scavengers or blocking ROS-dependent NF-kB activation significantly decreased ketone-dependent BDNF-upregulation in neurons, suggesting that ROS may have increased BDNF expressions to improve mitochondrial respiration as adaptive responses.
Conclusions: 3OHB initially generates ROS and poses oxidative stress. However, ROS appears to trigger adaptive responses against oxidative stress by upregulating BDNF through NF-kB activation, which can improve mitochondrial oxidative capacity and ultimately enhance neuroprotection
BHB/KD promotes PKA/CREB activation 
Another clever way to change the function/expression of multiple genes in one single step is to use a protein kinase.  Up to 30% of all human proteins may be modified by kinase activity.  
A protein kinase is an enzyme that modifies other proteins by chemically adding phosphate groups to them (phosphorylation). Phosphorylation usually results in a functional change of the target protein.
In the autism research you may well have come across PKA, PKB (Akt) and PKC. They clearly are disturbed in much autism.
The research shows that BHB activates PKA.
If you want good myelination you need PKA.
This might be another reason why BHB/KD is helpful in people with Multiple Sclerosis.
In much autism the myelin coating is found to be abnormally thin. 

BHB, Microglial Ramification and Depression (yes, depression)
I am increasingly impressed by research from China. The paper below by Chao Huang et al is excellent and I think we need a Chinese on the Dean’s List of this blog, it looks like he is the first.
Nantong, China on the Yangtze River and home to Chao Huang and more than 7 million other people 
Source: Wikipedia Dolly 442

The ketone body metabolite β-hydroxybutyrate induces an antidepression-associated ramification of microglia via HDACs inhibition-triggered Akt-small RhoGTPase activation. 


Abstract


Direct induction of macrophage ramification has been shown to promote an alternative (M2) polarization, suggesting that the ramified morphology may determine the function of immune cells. The ketone body metabolite β-hydroxybutyrate (BHB) elevated in conditions including fasting and low-carbohydrate ketogenic diet (KD) can reduce neuroinflammation. However, how exactly BHB impacts microglia remains unclear. We report that BHB as well as its producing stimuli fasting and KD induced obvious ramifications of murine microglia in basal and inflammatory conditions in a reversible manner, and these ramifications were accompanied with microglial profile toward M2 polarization and phagocytosis. The protein kinase B (Akt)-small RhoGTPase axis was found to mediate the effect of BHB on microglial shape change, as (i) BHB activated the microglial small RhoGTPase (Rac1, Cdc42) and Akt; (ii) Akt and Rac1-Cdc42 inhibition abolished the pro-ramification effect of BHB; (iii) Akt inhibition prevented the activation of Rac1-Cdc42 induced by BHB treatment. Incubation of microglia with other classical histone deacetylases (HDACs) inhibitors, but not G protein-coupled receptor 109a (GPR109a) activators, also induced microglial ramification and Akt activation, suggesting that the BHB-induced ramification of microglia may be triggered by HDACs inhibition. Functionally, Akt inhibition was found to abrogate the effects of BHB on microglial polarization and phagocytosis. In neuroinflammatory models induced by lipopolysaccharide (LPS) or chronic unpredictable stress (CUS), BHB prevented the microglial process retraction and depressive-like behaviors, and these effects were abolished by Akt inhibition. Our findings for the first time showed that BHB exerts anti-inflammatory actions via promotion of microglial ramification. 



NOTE:  Ramified Microglia = Resting Microglia


The brain microglia play important roles in sensing even subtle variations of their milieu. Upon moderate activation, they control brain activity via phagocytosis of cell debris and production of pro-inflammatory mediators and reactive oxygen species. However, a persistent activation would make the microglia transfer into a status with an amoeboid morphology tightly associated with neuronal damage and pro-inflammatory cytokine overproduction.

Unlike the activated microglia, the un-stimulated microglia are in a ramified status with extensively branched processes, an contribute to brain homeostasis via regulation of synaptic remodeling and neurotransmission. The ramified microglia has been shown to be associated with the induction of M2 polarization. A study by McWhorter et al. showed that elongation of macrophage by control of cell shape directly increases the expression of M2 markers and reduces the secretion of proinflammatory cytokines, suggesting that induction of microglial ramification may be a mechanism for regulation of microglial function. Methods that trigger microglial ramification may help treat brain disorders associated with neuroinflammation.
In this study, we found that BHB induces a functional ramification of murine microglia in both basal and inflammatory conditions in vitro and in vivo. The pro-ramification effects of BHB are associated with the change in microglial polarization and phagocytosis as well as the antidepressant-like effects of BHB in LPS- or chronic unpredictable stress (CUS)-stimulated mice. The ramified morphology in microglia is also induced by two BHB-producing stimuli fasting and KD, as well as two other HDACs inhibitors valproic acid (VPA) and trichostatin A (TSA). Given that microglial overactivation can mediate the pathogenesis of depression, induction of microglial ramification by BHB may have therapeutic significance in depression. 

These data confirm that BHB has an ability to transform the activated microglia back to their ramified and resting status in inflammatory conditions.

Recall the recent post about BHB and the Niacin Receptor HCA2/GPR109A in Autism:

The Chinese paper continues:

It is HDACs inhibition but not GPR109A activation that mediates the pro-ramification effect of BHB in microglia Akt inhibition abrogates the effects of BHB on microglial ramification, polarization, and phagocytosis
Akt inhibition prevents the antidepressant-like effects of BHB in acute and chronic depression models

Note that Akt is another name for Protein Kinase B (PKB)

One of the major findings in the present study is that the ketone body metabolite BHB as well as its producing stimuli fasting and KD induced reversible ramifications of murine microglia in vitro and in vivo, and these ramifications were not altered by pro-inflammatory stimuli. The ramified morphology induced by BHB seems to be a signal upstream of microglial polarization, and may mediate the antidepressant-like effect of BHB in depression induced by neuroinflammatory stimuli. Since the regulating effect of BHB in disorders associated with neuroinflammation has been well-documented, our findings provide a novel mechanism for the explanation of the neuroprotective effect of BHB in neurodegenerative and neuropsychiatric disorders from the aspect of the feedback regulation of microglial function by microglial ramification.
Induction of microglial ramification, a strategy neglected by most scientists for a long time, may have more important therapeutic significance than that of regulation of microglial polarization alone at the molecular level.

In experiments in vivo, we showed that BHB ameliorated the depressive-like behaviors induced by two neuroinflammatory stimuli LPS and CUS. These results are in accordance with previous reports, which showed that the BHB-producing stimuli, caloric restriction and fasting, produce potential antidepressant-like activities in both animals and humans. Thus, together with the pro-ramification effect of BHB in microglia in vitro, we speculate that the microglial shape change may be an independent signal that determines microglial function.

Our further analysis showed that the BHB-induced microglial ramification was mediated by the Rac1-Cdc42 signal, as BHB markedly increased the activity of Rac1 and Cdc42, and Rac1/Cdc42 inhibition attenuated the pro-ramification effect of BHB. The PI3K-Akt signal has been shown to mediate the activation of Rac1/Cdc42, and once accepting the signal from Akt, the Rac1-Cdc42 will be mobilized to promote lamellipodia/filopodia formation and cell shape change (Huang et al., 2016a). We showed that the BHB-induced microglial ramification was mediated by the Akt signal, as Akt inhibition suppressed the induction of microglial ramification by BHB. As a functional evidence for the involvement of Akt in the pro-ramification effect of BHB, Akt inhibition was found to block the functional changes in BHB-treated microglia in vitro and in vivo, including blockage of the anti-inflammatory and prophagocytic activity of BHB and abrogation of the antidepressant-like effects of BHB. Since the ramified morphology determines the anti-inflammatory phenotype in macrophages (McWhorter et al., 2013), our data suggest that there may exist a causal relationship between the ramified morphology and microglial function after BHB treatment, and this relationship may evidence the clinical significance of our findings, as the microglial process retraction has been shown to mediate the development of neurodegenerative and neuropsychiatric disorders.

Furthermore, considering the serum level of BHB in humans begin to rise to 6 to 8 mM with prolonged fasting (Cahill, 2006), investigation of whether the pro-ramification effect of BHB exists in human individuals should be of great value for the application of BHB in disease therapy. 


 Exposure to hypobaric hypoxia causes neuron cell damage, resulting in impaired cognitive function. Effective interventions to antagonize hypobaric hypoxia-induced memory impairment are in urgent need. Ketogenic diet (KD) has been successfully used to treat drug-resistant epilepsy and improves cognitive behaviors in epilepsy patients and other pathophysiological animal models. In the present study, we aimed to explore the potential beneficial effects of a KD on memory impairment caused by hypobaric hypoxia and the underlying possible mechanisms. We showed that the KD recipe used was ketogenic and increased plasma levels of ketone bodies, especially β-hydroxybutyrate. The results of the behavior tests showed that the KD did not affect general locomotor activity but obviously promoted spatial learning. Moreover, the KD significantly improved the spatial memory impairment caused by hypobaric hypoxia (simulated altitude of 6000 m, 24 h). In addition, the improving-effect of KD was mimicked by intraperitoneal injection of BHB. The western blot and immunohistochemistry results showed that KD treatment not only increased the acetylated levels of histone H3 and histone H4 compared to that of the control group but also antagonized the decrease in the acetylated histone H3 and H4 when exposed to hypobaric hypoxia. Furthermore, KD-hypoxia treatment also promoted PKA/CREB activation and BDNF protein expression compared to the effects of hypoxia alone. These results demonstrated that KD is a promising strategy to improve spatial memory impairment caused by hypobaric hypoxia, in which increased modification of histone acetylation plays an important role

Exogenous BHB prevents spatial memory impairment induced by hypobaric hypoxia

To further verify whether ketone body, a product of KD, has direct improving effect, we chose the most stable physiologic ketone body, BHB, for the subsequent experiment. In order to mimic the effect of KD as above described, the rats were pre-treated with BHB (at a dose of 200mg/kg/day) for 2 weeks and then submitted to Morris water maze test. Since intraperitoneal injection would allow substances to be absorbed at a slower rate and intraperitoneal injection would produce marginal effect during behavioral tests [16], we used the intraperitoneal injection of BHB, which has been applied in published reports [17, 18]. Although the rats in the control and BHB groups learned to find the platform with the same pattern during 5 days of acquisition training (Fig 4B), BHB could significantly improve the memory impairment induced by hypobaric hypoxia, represented by more crossing number, more time in the target quadrant, and decreased latency to first entry to platform compared to hypobaric hypoxia treatment alone (Fig 4C–4F). These results demonstrated that BHB has a direct memory-improving effect and served as the main executor of KD beneficial effects.

KD increases histone acetylation modification in the hippocampus

A previous study found that BHB is an endogenous HDAC inhibitor, and the KD recipe in our study substantially increased plasma levels of BHB. Then, we detected the effect of KD on histone acetylation in the hippocampus, which is responsible for learning and memory. As shown in Fig 5, the acetylated histone H3 (K9/K14), acetylated histone H3 (K14), and acetylated histone H4 (K12), were all increased in the hippocampus of the KD rats. Although the histone acetylation modifications listed above are decreased in hypoxia-treated rats, KD treatment could reverse the decreased levels of histone acetylation. The same pattern was displayed in the immunohistochemical staining, in which the hypoxia-induced decrease in acetylated histone H3 and acetylated histone H4 in the CA1 region of the hippocampus was reversed by KD treatment  

KD activates PKA/CREB signaling in the hippocampus

To explore a possible underlying mechanism of the beneficial effect of KD treatment on cognition, the activity of the PKA/CREB pathway in the four groups was also evaluated by western blot (Fig 7A). KD treatment was shown to not only increase the levels of PKA substrates and p-CREB (KD vs STD) but also reverse the decline in PKA substrates, p-CREB and CREB (KD-Hy vs STD-Hy). Although KD pre-treatment produced a partial restoration of PKA activity, p-CREB is nearly completely restore to its basic levels, which is may be account for its other upstream kinases, like calmodulin-dependent kinases [19]. Interestingly, the hypoxia-induced down-regulation of BDNF, a well-known neurotrophic factor involved in learning and memory formation processes, was also up-reregulated by KD treatment. These results demonstrated that KD treatment promoted PKA/CREB activation and BDNF protein expression. In order to detect whether KD promoted BDNF expression at mRNA levels, qRT-PCR assays were performed using BDNF specific primers. We found that KD-pretreatment significantly increased mRNA levels compared with that in hypobaric hypoxia group (Fig 7B). Next, we used ChIP-PCR to test if there might be increased enrichment of acetylated histones on the promoter of BDNF gene. We focused on the promoter I of BDNF gene, which response to neuronal activity [20). ]. The results showed that there is increased binding of acetylated histone H3 to the promoter I of BDNF gene (Fig 7C   

Concentrations of acetyl–coenzyme A and nicotinamide adenine dinucleotide (NAD+) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-β-hydroxybutyrate (βOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous βOHB, or fasting or calorie restriction, two conditions associated with increased βOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by βOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with βOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with βOHB conferred substantial protection against oxidative stress. 
Abnormalities in mitochondrial function and epigenetic regulation are thought to be instrumental in Huntington's disease (HD), a fatal genetic disorder caused by an expanded polyglutamine track in the protein huntingtin. Given the lack of effective therapies for HD, we sought to assess the neuroprotective properties of the mitochondrial energizing ketone body, D-β-hydroxybutyrate (DβHB), in the 3-nitropropionic acid (3-NP) toxic and the R6/2 genetic model of HD. In mice treated with 3-NP, a complex II inhibitor, infusion of DβHB attenuates motor deficits, striatal lesions, and microgliosis in this model of toxin induced-striatal neurodegeneration. In transgenic R6/2 mice, infusion of DβHB extends life span, attenuates motor deficits, and prevents striatal histone deacetylation. In PC12 cells with inducible expression of mutant huntingtin protein, we further demonstrate that DβHB prevents histone deacetylation via a mechanism independent of its mitochondrial effects and independent of histone deacetylase inhibition. These pre-clinical findings suggest that by simultaneously targeting the mitochondrial and the epigenetic abnormalities associated with mutant huntingtin, DβHB may be a valuable therapeutic agent for HD.  

Conclusion
At the end of this fifth post on ketones and autism, I think we have established beyond any doubt that ketones can do some amazing things for numerous dysfunctions and diseases.
The question remains how much you need to achieve the various possible benefits. 
The next question, already put to me by one parent, is how do you measure such a benefit.  Some people’s idea of treating autism is just to eradicate disturbing behaviours like SIB and ensure a placid, cooperative child when out in public.  Other people notice small cognitive and speech changes, because they spend hours a day teaching their child. Small but significant cognitive improvement may not show up on autism rating scales.
You would expect a dose dependent response, so the more ketones the bigger the response, which suggests that the full Ketogenic Diet (KD) is the ultimate option.
A lot does seem to be possible just with BHB and C8 (caprylic acid) as supplements to a regular diet.
Adults with Alzheimer’s, or Huntington’s, or Multiple Sclerosis (MS) all stand to potentially benefit from ketone supplements.
Children/adults with certain single-gene autisms, not limited to Kabuki and Pitt Hopkins potentially should benefit from ketone supplements.
Interestingly, another benefit of BHB is on mood; it seems to make some people just feel much better, apparently all due to the effect on microglia. So perhaps autism parents who take antidepressants should try BHB instead.







Thursday 26 July 2018

Promoting Spontaneous Speech in Autism – Behavioral Therapies and/or BHB?


One key issue for most people with more severe autism (DSM5 level 3, any DSM3, or just SDA – Strictly Defined Autism) is getting them to fully use their capacity to communicate.
Many such people do learn to talk, but often this is a very matter of fact kind of speech, that is limited to answering questions and making requests.
Often the limiting factor is not vocabulary, grammar or vocalization. Many can put ideas in words on paper, they can sing and read aloud; but something is lacking when it comes to conversation.
You can use behavioral therapy (like Verbal Behavior, VB) and some of the more relevant parts of speech therapy to encourage more extensive speech, but it is a real slog. When you spend less time on 1:1 “speech training”, in order to develop academic school work, you end up with less speech.
I was discussing this with Monty’s assistants, the need to take a step back and refocus on speech as a skill, in addition to regular school work.  It is good to be able to master algebra, but in any social situation communication remains number one.
I have long wondered if it is a behavioural problem, a structural brain problem or a treatable biological problem. I do see parallels with how typical people speak foreign languages. For example, if I was on a train in Germany and somebody wanted to talk to me in German, I would keep my answers short and simple; I would not be trying to keep the conversation going. My German is very rusty, but I can read it aloud and yes, I could sing in it. I would not make small talk in German.
I do consider people like Monty, now aged 15 with autism, not to have a first language; silence is their first language and their mother tongue is like a second language. Some readers of this blog are exceptions, but most people are pretty weak in their second language. For them it never becomes intuitive, you have to painfully learn what preposition takes what case and how to decline nouns and conjugate verbs. You can make a strong case that people who do not begin to speak until 4 years old have missed a critical window in how the brain develops and so when language does slowly begin to come, it can never become truly fluent, a bit like my German.
I do have a broader interest in how you acquire language. Monty's big brother is bilingual and also pretty fluent in Russian and German and chatty in French. This all came with minimal effort, but being exposed from birth to two languages and then learning more languages in the conventional way.  
Many readers of autism forums ask about what pill can you take to promote speech. I always thought this was rather wishful thinking, in that you cannot target such a specific aspect of someone’s autism. Just like there may be no magic pill for algebra.

Writing before talking?
One idea I had to promote more prolonged “conversation” was to first have Monty write about the subject.  I agreed with Monty’s assistants one new exercise, which is to have him write a daily diary of his day and then later on have him retell the day’s events, but without reference to the diary.
It does indeed work, having written about the subject, when later asked to talk about it, there is a much longer and more detailed conversation.
No pills required.

The Ketone BHB
The reason for all the recent posts in this blog about ketones and autism is that perhaps there actually is a “pill” you can take to promote speech.
Our reader Agnieszka in Poland has been experimenting with ketones for some time and since her son responds to most of the things in Monty’s PolyPill, I assume that there is a good chance that anything new that works for her son might also work for Monty.
One area that the ketone BHB seems to help in Agnieszka’s case is promoting speech and BHB does indeed have the very same effect in Monty.
One effect of sulforaphane/broccoli was increased verbalization. If your child is four years old, increased verbalization would be something to celebrate, but by 15 years old you want relevant speech (for some people sulforaphane does indeed produce increased relevant speech).  Very encouragingly, BHB seems to deliver an increase in intelligent, relevant, spontaneous speech. These are things that Monty could write but would not say, unprompted.
There are big gaps in the scientific data about ketone supplements in humans. These supplements are only widely available in North America, even though it looks like most commercial products are repackaged from Chinese bulk chemical producers.
It appears that the most effective therapy is a combination of a precursor to ketones (C8) and a salt of the BHB ketone (Sodium, Calcium, Magnesium or Potassium Beta-Hydroxybutyrate).
According to one Chinese bulk producer, the Calcium salt of BHB is not very effective, they recommend Sodium and/or Magnesium.
By using a salt of BHB, you are going to consume significant amounts of sodium, calcium, magnesium or potassium. This may be unwise for some people.
The objective is to raise the amount of BHB in your bloodstream.
You can measure BHB in urine inexpensively, but the more reliable blood testing equipment is more expensive.
The study below evaluated one widely available commercial supplement:- 


Figure 1b: mean, standard deviation of D-βHB (mmol/L) level in serum of all subjects () within a time period of 5.5 h after intake of βHB salt mixture (0.5 g/kg BW). 

The supplement used was Ketosports KetoCaNa Orange.
In fact the dose of Ca/Na Betahydroxybutyrate was very high, 0.5g per Kg. In the case of a typical adult that might be 40g per day.
That would contain:-

·      23g of BHB

·      2.6g of sodium

·      2.3g of calcium

That is quite a lot of sodium and calcium.
Some products are exclusively Potassium Betahydroxybutyrate, in those for each 23g of BHB you would get 8,700 mg of potassium, which is way too much to take at once. I am amazed it has not been banned.
When it comes to data on the use of C8 MCT oil in humans to produce BHB, we have the following study. The chart they produced is the total of BHB and another ketone, acetoacetate (AcAc), but we can extract the data on BHB itself. 


Results: C8 was the most ketogenic test oil with a day-long mean ± SEM of +295 ± 155 µmol/L above the CTL. C8 alone induced the highest plasma ketones expressed as the areas under the curve (AUCs) for 0–4 and 4–8 h (780 ± 426 µmol h/L and 1876 ± 772 µmol h/L, respectively); these values were 813% and 870% higher than CTL values (P < 0.01). CO plasma ketones peaked at +200 µmol/L, or 25% of the C8 ketone peak. The acetoacetate-to-β-HB ratio increased 56% more after CO than after C8 after both doses.

Conclusions: In healthy adults, C8 alone had the highest net ketogenic effect over 8 h, but induced only half the increase in the acetoacetate-to-β-HB ratio compared with CO. Optimizing the type of MCT may help in developing ketogenic supplements designed to counteract deteriorating brain glucose uptake associated with aging. This trial was registered at clinicaltrials.gov as NCT 02679222.




Plasma concentration and summed daily means (far right) during the metabolic study days for total ketones (β-HB and AcAc) obtained without an added test oil (CTL; ●) or after taking two 20-mL doses of CO alone (), C10 alone (□), medium-chain TGs (C8-C10; ), or C8 alone (). The open arrow indicates when the breakfast plus test oil was consumed; the solid arrow indicates when the test oil alone was consumed without an accompanying meal at midday. Data for metabolic study days on which CO+C8-C10 and CO+C8 were tested are not shown here for clarity, but their AUC data are shown in Figure 2. Values are means ± SEMs; n = 9/point. *Different from CTL, P < 0.05. AcAc, acetoacetate; CO, coconut oil; CTL, control; C8, tricaprylin; C10, tricaprin; β-HB, β-hydroxybutyrate. 

Our 2-dose test protocol (breakfast and midday) generated 2 peaks of plasma total ketones throughout 8 h, with the second dose inducing 3.5 and 2.4 times higher ketones with C8 than with CO, respectively. The first dose taken with a meal would be a more typical pattern but resulted in less ketosis that without a meal. One limitation of this study design is that the metabolic study period was only 8 h. A longer-term study lasting several weeks to months would be useful to assess the impact of regular MCT supplementation on ketone metabolism.

In summary, C8 was the most ketogenic MCT tested in this acute 8-h study and its ketogenic effect was significantly higher in the absence of an accompanying meal. Despite a low net ketogenic effect, CO may still be of interest because of its effect on plasma acetoacetate-to-β-HB ratio. With the help of positron emission tomographic imaging and the ketone tracer 11C-acetoacetate (2, 18, 20), it is now possible to investigate the impact on tissue ketone uptake of various ketogenic interventions.



Areas Under the Curve = AUC 

Plasma concentration and summed means of 0- to 4-h and 4- to 8-h AUCs for plasma total ketones (i.e., AcAc and β-HB combined) (A) and for the mean AcAc-to-β-HB ratio (B). Bars represent no test oil consumed (CTL) or values after taking 2 doses of CO alone, C10 alone, medium-chain TGs (C8-C10), C8 alone, CO+C8-C10 (50:50), or CO+C8 (50:50). Values are means ± SEMs; n = 9. The AUC for 0–4 h was significantly different from the AUC for 4–8 h under all conditions. Labeled means without a common letter differ (a < b < c < d < e and A < B < C < D < E), P < 0.05. AcAc, acetoacetate; CO, coconut oil; CTL, control; C8, tricaprylin; C10, tricaprin; β-HB, β-hydroxybutyrate.

If we assume AcAc/BHB from C8 oil is 0.8 and that taking C8 without foode gives a total peak ketone (AcAc + BHB) of 0.5 mmol/L in blood. That implies we can approximate peak BHB as 0.28 mmol/L and peak AcAc as 0.22 mmol/L.
The jumbo dose of 23g of BHB produced peak BHB of 0.6 mmol/L in adults.
If the BHB level in blood is linearly related to the dose of BHB supplement, the we might assume that 15ml of Ketoforce produces 0.15 mmol/L (3.9*1.5/23*0.6).
If the Chinese are right that calcium BHB is not effective, then 15ml of Ketoforce likely produces a bit more than 0.15mmol/L., since it contains sodium BHB and potassium BHB.
So we might assume that my 20ml of C8 and 15ml of Ketoforce would produce  a peak BHB in the bloodstream of about 0.5 mmol in a 55kg boy and that slightly more is coming from the C8 than the BHB salt.
As you can see from the chart 0.5 is not very much and just at the lower edge of nutritional ketosis. With supplementation 0.5 is the peak level; it will rapidly fall back to the starting level.


So via supplementation we have a brief period of mild nutritional ketosis.

Anyone who has done their homework on Ketones will have come across Dominic D’Agostino.  He is a researcher with a big interest in ketones. He has published interesting research and has his own blog on the subject.

I saw his advice that suggested starting with 10ml of C8 and 10ml of KetoForce.
The producer suggests 30ml a day of Ketoforce in adults.
10 ml of Ketoforce contains
·      3.9 g of BHB

·      533 mg of Potassium

·      533 mg of Sodium

Even 500mg of potassium is going increase potassium levels in your blood.
If you happen to be taking bumetanide for autism, you will be losing potassium and likely taking a potassium supplement. Depending on your bumetanide dosage and potassium supplement, you may well be able to make some adjustments and cope with 10 ml of Ketoforce. 

Speech and C8/Ketoforce Dosage
Our reader Agnieszka was very scientific and tried different BHB supplements and measured  BHB in urine. She found Ketoforce the most effective at producing BHB in urine; speech was one big area of improvement, but not the only one.
I took the advice of Dr D’Agostino, the ketone guru, and combined Ketoforce with C8 in my experiment.
Starting with 10ml of C8 and 10ml Ketoforce, it did produce a marginal change in Monty, but increasing to 20ml of C8 and 15ml of Ketoforce produced a clear increase in spontaneous speech.

Work in progress
Clearly this is a work in progress. Ideally I would want to get all the BHB from C8, since then I do not need to worry about sodium and potassium.
Ketoforce is pretty expensive in the US and very expensive in Europe. C8 is not cheap, but much more reasonable.
Other MCT oils and coconut oil may be cheaper, but are very much less potent, so C8 is the most cost effective MCT oil to produce the ketone BHB.
A naturopathic physician in the US, called Dr Bruce Fife, has written extensively about “reversing autism” with coconut oil; that kind of language will make many people wary. He does suggest the mode of action is calming microglia, which is something BHB should be doing. Regular coconut oil will produce BHB, but you would have to eat a great deal of it.  Coconut oil it is not cheap and so it is more cost effective to use C8 oil. The effect of coconut oil (CO) was shown earlier on in this post, in the above line graph, where the triangle represents coconut oil.
Coconut oil, counter intuitively, actually lowers your blood cholesterol, so it actually is a healthy oil, but if it is the ketone BHB you are after, it does not look the best choice.
It is possible that coconut oil does something clever that is unrelated to BHB.

Underlying Mode of Action
By investigating all the modes of action of BHB, this may lead to a more effective therapy. BHB is a signalling molecule and if you know which of its many effects is the key one, you may find an alternative signalling molecule that gives a more potent result.
I have a good effect from C8/Ketoforce, but I would like more of the same; but without the full ketogenic diet and not causing a problem with excess sodium, potassium, calcium or magnesium.

In the coming posts we will look into BHB's other modes of action. It will get quite interesting and we will see how one might even treat psoriasis and Multiple Sclerosis with BHB, because it is an activator of the niacin receptor HCA2. There is a potent HCA2 agonist drug, dimethyl fumarate.