Showing posts with label insulin. Show all posts
Showing posts with label insulin. Show all posts

Thursday 19 July 2018

Ketones and Autism Part 2 - Ketones as a Brain Fuel to treat Alzheimer’s, GLUT1 Deficiency and perhaps more

Today’s post looks at the role ketones can play as a fuel for the brain.

The research has already shown that in young babies there is insufficient glucose to fuel their power-hungry growing brains and so ketones provide up to 40% of the fuel to their brains.
Glucose or Ketones at the pump?

This does show any sceptics that you can indeed safely combine two sources of fuel at the same time in humans; we have all done it.
This process works in tiny babies because their diet is rich in medium chain fatty acids, which become the ketones.
Only mitochondria in your brain and your muscles can be fuelled by ketones; some elite athletes take advantage of this.
People who are overweight have excess adipose tissue (fat) and when in ketosis, fatty acids from this tissue are released into your blood and travel to the liver where they produce ketones. Mitochondria can also burn fatty acids directly. People losing weight on the ketogenic diet are burning fat and ketones as their main fuel source. To lose weight you do have to be in calorie deficit, you cannot just eat unlimited fat.
Athletes want to improve their performance and some use ketones to achieve this. The fat they are burning is from diet, not from accumulated over-eating.
Ketones as a brain fuel is a niche subject, but a growing one.

Low brain glucose uptake
Low brain glucose uptake is a feature or Alzheimer’s disease and also of a rare inborn condition called GLUT1 deficiency, which appears as epilepsy, MR/ID and with features of autism. Infants with GLUT1 deficiency syndrome have a normal head size at birth, but growth of the brain and skull is slow, in severe cases resulting in an abnormally small head size (microcephaly).

GLUT1 (glucose transporter 1) occurs in almost all tissues, with the degree of expression typically correlating with the rate of cellular glucose metabolism. It is expressed in the endothelial cells of barrier tissues such as the blood brain barrier.
Glucose delivery and utilization in the human brain is mediated primarily by GLUT1 in the blood–brain barrier and GLUT3 in neurons.
GLUT3 is most known for its specific expression in neurons and was originally designated as the neuronal glucose transporter.
GLUT4 is the insulin-regulated glucose transporter found primarily in adipose tissues (fat) and striated muscle (skeletal and cardiac), but also in the brain.
So, in neurological disorders it is important to optimize GLUT1, GLUT3, GLUT4 and insulin.  In GLUT1 deficiency, as the name suggests, there is an inadequate supply of glucose crossing the blood brain barrier. In people with insulin resistance (T2 diabetes, Alzheimer’s etc) GLUT4 may be impaired.

Insulin resistance in the brain
Insulin resistance in the brain is highly complex and only partially understood; but it does lead to numerous problems. Glucose in the blood does not get taken up adequately into neurons which then become starved of fuel. We will see how this can be overcome by reverting to ketones as an alternative fuel.
At this point I digress a little into the detail of insulin resistance and glucose transport.                                                                             

Insulin resistance is a condition in which cells fail to respond normally to the hormone insulin. The body produces insulin when glucose starts to be released into the bloodstream from the digestion of carbohydrates  in diet. Under normal conditions of insulin reactivity, this insulin response triggers glucose being taken into body cells, to be used for energy, and inhibits the body from using fat for energy, thereby causing the concentration of glucose in the blood to decrease as a result, staying within the normal range even when a large amount of carbohydrates is consumed. During insulin resistance, excess glucose is not sufficiently absorbed by cells, even in the presence of insulin, causing an increase in the level of blood sugar.

The following paper is very interesting, if you can access the full text version

Considerable overlap has been identified in the risk factors, comorbidities and putative pathophysiological mechanisms of Alzheimer disease and related dementias (ADRDs) and type 2 diabetes mellitus (T2DM), two of the most pressing epidemics of our time. Much is known about the biology of each condition, but whether T2DM and ADRDs are parallel phenomena arising from coincidental roots in ageing or synergistic diseases linked by vicious pathophysiological cycles remains unclear. Insulin resistance is a core feature of T2DM and is emerging as a potentially important feature of ADRDs. Here, we review key observations and experimental data on insulin signalling in the brain, highlighting its actions in neurons and glia. In addition, we define the concept of 'brain insulin resistance' and review the growing, although still inconsistent, literature concerning cognitive impairment and neuropathological abnormalities in T2DM, obesity and insulin resistance. Lastly, we review evidence of intrinsic brain insulin resistance in ADRDs. By expanding our understanding of the overlapping mechanisms of these conditions, we hope to accelerate the rational development of preventive, disease-modifying and symptomatic treatments for cognitive dysfunction in T2DM and ADRDs alike. 

Sources of insulin in the brain. Insulin levels in cerebrospinal fluid (CSF) are much lower than in plasma but these levels are correlated, indicating that most insulin in the brain derives from circulating pancreatic insulin. Insulin enters the brain primarily via selective, saturable transport across the capillary endothelial cells of the blood–brain barrier (BBB).
Despite glucose being the major energy source for the brain, the uptake, transport and utilization of glucose in neurons is only influenced by insulin and is not dependent on it
The insulinindependent glucose transporter GLUT3 is the major glucose transporter in neurons and is present in very few other cell types in the body. The density and distribution of GLUT3 in axons, dendrites and neuronal soma correlates with local cerebral energy demands. Insulin is not required for GLUT3mediated glucose transport; instead, NMDA receptormediated depolarization stimulates consumption of glucose, which prompts glucose uptake and utilization via GLUT3. 
Although most glucose uptake in neurons occurs via GLUT3, insulinregulated GLUT4 is also coexpressed with GLUT3 in brain regions related to cognitive behaviours — at least in rodents. These regions include the basal forebrain, hippocampus, amygdala and, to lesser degrees, the cerebral cortex and cerebellum. 
Activation by insulin induces GLUT4 translocation to the neuron cell membrane via an AKTdependent mechanism and is thought to improve glucose flux into neurons during periods of high metabolic demand, such as during learning. Interestingly, GLUT4 is also expressed in the hypothalamus, a key area for metabolic control. Deletion of GLUT4 from the CNS in mice results in impaired glucose sensing and tolerance, which might be due in part to an absence of GLUT4 in the hypothalamus.

Brain insulin resistance definition. Insulin resistance in T2DM has been defined as “reduced sensitivity in body tissues to the action of insulin”. Similarly, brain insulin resistance can be defined as the failure of brain cells to respond to insulin. Mechanistically, this lack of response could be due to downregulation of insulin receptors, an inability of insulin receptors to bind insulin or faulty activation of the insulin signalling cascade. At the cellular level, this dysfunction might manifest as the impairment of neuroplasticity, receptor regulation or neurotransmitter release in neurons, or the impairment of processes more directly implicated in insulin metabolism, such as neuronal glucose uptake in neurons expressing GLUT4, or homeostatic or inflammatory responses to insulin. Functionally, brain insulin resistance can manifest as an impaired ability to regulate metabolism — in either the brain or periphery — or impaired cognition and mood 

Studies have yet to show whether T2DMassociated cognitive impairment and brain neuroimaging findings are a consequence of brain insulin resistance or are due to other factors that cooccur with systemic insulin resistance. Common comorbidities of systemic insulin resistance in T2DM — such as hyperglycaemia, advanced glycation end products, oxidatively dam aged proteins and lipids, inflammation, dyslipidaemia, athero sclerosis and microvascular disease, renal failure and hypertension — all have their own complex effects on brain function through a variety of mechanisms independent of insulin signalling. Furthermore, evidence suggests that systemic insulin resistance or high circulating levels of insulin affects the function of the BBB by downregulating endothelial insulin receptors and thus decreasing permeability of the BBB to insulin. This change in permeability is potentially of great importance as it could lead to decreased brain insulin levels and decreased insulinfacilitated neural and glial activity40. On the other hand, T2DM can lead to damage of the BBB, which results in increased permeability to a variety of substances

Brain insulin resistance in ADRDs
• Increasing age is associated with decreasing cortical insulin concentration and receptor binding in older adults without dementia 
•Brain tissue from those with Alzheimer disease (AD) shows major abnormalities in insulin signalling, including - Decreased insulin, insulin receptor and insulin receptor substrate 1 (IRS1) mRNA and/or protein expression levels
Decreased activation of insulin pathway molecules (for example, IRS1 and AKT) with ex vivo stimulation
Increased basal phosphorylation levels of multiple insulin–IRS1–AKT pathway molecules
 Positive correlation between phosphorylated IRS1 and other pathway molecules and AD pathology 
• Intranasal insulin administration improves cognitive functioning in humans with AD or mild cognitive impairment and improves measures of insulin signalling, amyloid-β and cognitive behaviours in AD model mice 
Brain insulin resistance might be a feature of other neurodegenerative diseases

Insulin receptor expression is decreased and AKT signalling is abnormal in the substantia nigra in Parkinson disease
Abnormal phosphorylated IRS1 expression is observed in tauopathies but is not seen in synucleinopathies or TDP-43 proteinopathies
Aside from treatment with insulin itself, insulinsensitizing medicines commonly used in T2DM have attracted growing interest as potential therapies for brain insulin resistance in ADRD. For instance, investigators have begun testing of metformin, the most commonly prescribed drug for T2DM, in nondiabetic individuals with MCI or early dementia due to AD, with some signs of benefit. In addition, thiazolidinedionebased nuclear peroxisome proliferatoractivated receptorγ (PPARγ) agonists, which were originally developed as insulin sensitizers for T2DM, have shown numerous beneficial neural effects in animal models of neuro degenerative diseases

Autism and GLUT1 deficiency:

Another excellent paper:-  

Brain energy metabolism in Alzheimer’s disease (AD) is characterized mainly by temporo-parietal glucose hypometabolism. This pattern has been widely viewed as a consequence of the disease, i.e. deteriorating neuronal function leading to lower demand for glucose. This review will address deteriorating glucose metabolism as a problem specific to glucose and one that precedes AD. Hence, ketones and medium chain fatty acids (MCFA) could be an alternative source of energy for the aging brain that could compensate for low brain glucose uptake. MCFA in the form of dietary medium chain triglycerides (MCT) have a long history in clinical nutrition and are widely regarded as safe by government regulatory agencies. The importance of ketones in meeting the high energy and anabolic requirements of the infant brain suggest they may be able to contribute in the same way in the aging brain. Clinical studies suggest that ketogenesis from MCT may be able to bypass the increasing risk of insufficient glucose uptake or metabolism in the aging brain sufficiently to have positive effects on cognition.

Push-pull: two distinct strategies to supply the brain with energy substrates. Glucose is the brain’s main fuel and is taken up by the brain in relation to demand. Hence, this is a “pull” strategy because glucose is pulled into the cell following neuronal activation and the subsequent decrease in neuronal glucose concentrations. Ketones are the brain’s main alternate fuel to glucose and are taken up by the brain in relation to their presence in blood. Hence, this is a “push” strategy because ketones are pushed into the brain in direct proportion to their concentrations in the blood.

5 Cognitive benefits of increasing brain ketone supply

Since brain ketone uptake is still normal in mild to moderate AD and the problem of low brain glucose uptake appears to be contributing to declining cognition in AD, it is reasonable to hypothesize that providing the brain with more ketones may delay any further cognitive decline. This hypothesis has been supported by results from acute and chronic studies in AD patients and in the prodromal condition to AD – mild cognitive impairment. Other trials with ketogenic supplements in AD are ongoing. Conditions involving acute or long-term cognitive problems including post-insulin hypoglycemia and epilepsy also respond to a ketogenic diet or supplement.

One of the reasons that type 2 diabetes is such an important risk factor for AD may be due to insulin resistance. The brain has long been thought to function independently of insulin, but this is now being challenged. Insulin resistance not only affects glucose uptake by peripheral tissues but it also blocks ketogenesis, thereby limiting production of ketones to be taken up by the brain. Indeed, if the insulin resistance of type 2 diabetes in some way impairs brain glucose metabolism, brain energy supply is in fact in double jeopardy because insulin excess also blocks ketogenesis from long chain fatty acids stored in adipose tissue thereby restricted access not just of the brain’s primary fuel (glucose) but its main back-up fuel (ketones) as well. One potential solution is that ketogenesis from MCFA appears to be independent of insulin, in which case a ketogenic MCFA supplement should still be able to supply the brain with ketones despite the presence of insulin resistance or type 2 diabetes. This is an active area of research.  

6 Ketones and infant brain development

Raising plasma ketones is commonly viewed as risky, primarily because ketosis is associated with uncontrolled type 1 diabetes, i.e. an acute and severe absence of insulin. However, pathological ketosis needs to be distinguished from nutritional ketosis: the former is associated with metabolic ketoacidosis, i.e. plasma ketones exceeding 15 mM, which is medically serious condition requiring rapid treatment. In contrast, the latter is associated with plasma ketones below 5 mM and can be safely induced by short- or long-term dietary modification. The very high fat ketogenic diet induces nutritional, not pathological ketosis. It has been used for nearly 100 years as a standard-of-care for intractable childhood epilepsy and is rarely associated with serious side-effects despite producing plasma ketones averaging 2–5 mM for periods commonly exceeding 2 years. Its mechanism of action is still poorly understood but the efficacy of this dietary ketogenic treatment for intractable epilepsy is greater in younger infants suggesting a possible link the well-established but often overlooked importance of ketones in infant brain development.

During lactation, the human infant brain metabolises >50% of the fuel provided, despite the brain representing only 12–13% of body’s weight. Glucose supplies about 30% of the late term fetus’s brain energy requirements and about 50% of the neonate’s brain energy requirements; the difference is provided by ketones. Therefore, ketones are an obligate brain fuel during an infant’s development, as opposed to being an alternative brain fuel in the adult human, i.e. only needed when glucose is limiting. Ketones are more than just catabolic substrates (fuel) for the developing brain – they are also important anabolic substrates because they supply the majority of carbon used to synthesize brain lipids such as cholesterol and long chain saturated and monounsaturated fatty acids. 


Unique route of medium chain fatty acid (MCFA) absorption compared to other common long chain dietary fatty acids. The lymphatic and peripheral circulation1 distribute most common long chain fatty acids as chylomicrons throughout the body, whereas MCFA are mostly absorbed directly via the portal vein to the liver2  

MCFA are more rapidly absorbed from the gut directly to the liver via the portal vein compared to long chain fatty acids which are absorbed primarily via the lymphatic duct and into the peripheral circulation. MCFA are also more easily β-oxidized in mitochondria because they do not require activation to CoA esters by carnitine. Both the rapid absorption and β-oxidation of MCFA suggest these fatty acids have a physiologically important function. Theoretically, this function could include elongation to long-chain fatty acids but, in practice, is probably limited to ketogenesis, especially in infancy which is the only period when it is normal to be regularly consuming MCFA.

Long chain fatty acids are the main alternate fuel to glucose for most tissues. They can also be taken up by the brain but the reason they are not a useful fuel for the brain is because their rate of uptake is insufficient to meet the demand for energy once glucose becomes limiting. However, MCFA such as octanoate (caprylic acid) can be taken up rapidly and be metabolized by the brain. Whether MCFA have direct effects on the brain or are principally metabolized to ketones before exerting any effect as fuels, lipid substrates or lipid signalling molecules remains to be seen. 

Ketones for Alzheimer’s? AC1202/4 
A lot of money is being spent on developing variants of caprylic acid (C8) as a medical food to treat one feature of Alzheimer’s. This medical food market has even attracted Nestle, the Swiss chocolate to baby food giant, to invest in ketones.
Even though clinical trials have not yet been successfully completed, American doctors are already prescribing a product called Axona to people with Alzheimer’s.
It looks like there are plenty of sceptics, but it looks like plenty of people are paying $80 a month for their Axona (>95% C8 oil). One packet of Axona powder, contains 20 grams MCTs almost exclusively C8.
You can see from the clinicals trials that Accera have been comparing the effectiveness of generic (unpatentable) C8 vs their two proprietary powders called AC-1202 and AC-1204. Clearly Accerra want to maximize plasma BHB, but in a way that has patent protection.
Since C8 is not so expensive when bought in bulk, the obvious alternative is just to drink C8 and in the way that best promotes its absorption and the production of ketones, which would seem to be when you wake up and before you have eaten anything.

CNS therapeutics company Accera's AC-1204 has failed to demonstrate a positive outcome in the Phase III trial for the treatment of patients with mild-to-moderate Alzheimer's disease. 
AC-1204 is a small-molecule drug compound designed to leverage the physiological ketone system in order to address the deficient glucose metabolism in Alzheimer's. 
The ketones are thought to have a potential to restore and improve neuronal metabolism, resulting in better cognition and function.
The trial results indicated that the drug did not show a statistically significant difference at week 26 when compared with placebo, as measured by the Alzheimer's disease assessment scale-cognitive subscale test (ADAS-Cog). 
"The formulation of the drug was changed between the Phase II and Phase III studies."
The double-blind, randomised, placebo-controlled, parallel-group Phase III (NOURISH AD) trial evaluated the effects of daily administration of AC-1204 in the subjects for 26 weeks.
Accera research and development vice-president Samuel Henderson said: "The formulation of the drug was changed between the Phase II and Phase III studies. 
"Unfortunately, this change in formulation had the unintended consequence of lowering drug levels in patients. We are confident that our newly developed formulation will provide increased exposure and allow a more conclusive test of drug efficacy." 
The primary and key secondary endpoints of the trial are the measure of AC-1204 effects on memory, cognition and global function. 
While the drug was found to be safe with high levels of tolerability, a detailed pharmacokinetic analysis showed that the modified formulation used in the study led to a decrease in drug plasma levels when compared to prior formulations. 

FDA hit Accera with a warning letter in 2013 on the grounds its marketing materials caused Axona to be classed as a drug. Accera continues to market Axona as a medical food for Alzheimer’s but has tweaked its website since the warning letter.
Axona and AC-1204 both provide patients with a source of caprylic triglyceride—also known as fractionated coconut oil—that is intended to increase the availability of ketones to the brain. The potential of the therapeutic approach has enabled Accera to pull in more than $150 million from backers including Nestlé, according to SEC filings.

Ketones for GLUT1 deficiency?  C7 Triheptanoin
It looks like the star clinician/researcher for people with GLUT1 deficiency is Dr. Juan Pascual, Associate Professor of Neurology and Neurotherapeutics, Pediatrics, and Physiology at UT Southwestern Medical Center.
As we saw earlier you need the transporter GLUT1 for glucose to cross the blood brain barrier and then provide fuel for the mitochondria in the brain. 
It has been known for some time that people with GLUT1 deficiency make improvements on the ketogenic diet.  Now in the previous post we saw how the effect on epilepsy of the KD comes via a change in the mix of bacteria in the gut; this eventually leads to a sharp increase in the ratio of GABA/Glutamate in the brain. This reduces seizures, which are a feature of GLUT1 deficiency.
Dr Pascual wants a second benefit from the ketogenic diet, having got the benefit from the gut bacteria he wants to benefit from the ketones as a fuel, just like some Alzheimer’s researchers.
This time though he has picked another MCT (medium chained triglyceride) he picked C7.
C7 is not something you can pick up from your specialist ketone supplier. It is still very much a research chemical.
Dr Pascual did not start with C8 because he has done his homework.  He actually wants some help for his GLUT1 deficient patients from some C5 ketones and a good way to produce them is from C7.
Using C7 oil Dr Pascual is also going to produce BHB (beta-hydroxybutyrate) and acetoacetate, just like all those athletes, body builders, slimmers and older people with Alzheimer’s are doing with the KD, C8 and BHB.

           Metabolism of glucose, C7-derived heptanoate and 5-carbon (C5) ketones in the brain

Glial metabolism is distinct from neuronal metabolism. Glucose can access both glia (via GLUT1) and neurons (via GLUT3), fueling the TCA cycle (CAC). In glia, pyruvate is converted into oxaloacetate (OAA) via carboxylation, donating net carbon to the TCA cycle (anaplerosis). This reaction can be impaired in G1D. Like glucose, the C7 derivative heptanoate and related metabolites (i.e., the 5-carbon ketones beta-ketopentanoate and beta-hydroxypentanoate) also generate acetyl-coenzyme A (Ac-CoA) but, unlike the 4-carbon ketone bodies beta-hydroxybutyrate and acetoacetate, they can also be incorporated into succinyl-coenzyme A (Suc-CoA) via propionyl-CoA (Prop-CoA) formation, supplying net, anaplerotic carbon to the cycle. In addition to 5-carbon (C5) ketones, the 4-carbon ketone bodies beta-hydroxybutyrate and acetoacetate are also metabolites of C7.

Dr. Pascual led the JAMA study that relied on data from a worldwide registry he created in 2013 for Glut1 deficiency patients. The research tracked 181 patients for three years, finding that a modified Atkins diet that includes less fat and slightly more carbohydrates than the standard ketogenic diet helped reduce seizures and improved the patients' long-term health. The study also found earlier diagnosis and treatment of the disease improved their prognosis.
In addition, Dr. Pascual is overseeing national clinical trials that are testing whether triheptanoin (C7) oil improves the intellect of patients by providing their brains an alternative fuel to glucose. The trials will last five years and are funded with more than $3 million from the National Institutes of Health.

So far, the nearly 40,000 Americans potentially living with the disease have had only one primary option for treating symptoms: a high-fat, low-carbohydrate ketogenic diet that can limit seizures. The diet works in about two-thirds of patients but does not improve their intellect and carries long-term risks such as kidney stones and metabolic abnormalities.

Dr. Pascual expects the modified diet from the JAMA study and the C7 oil will prove at least as effective as the ketogenic diet in preventing seizures - without the health risks - while feeding the brain vital fuel to improve learning.

Background: Ketones are the brain's main alternative fuel to glucose. Dietary medium-chain triglyceride (MCT) supplements increase plasma ketones, but their ketogenic efficacy relative to coconut oil (CO) is not clear.

Objective: The aim was to compare the acute ketogenic effects of the following test oils in healthy adults: coconut oil [CO; 3% tricaprylin (C8), 5% tricaprin (C10)], classical MCT oil (C8-C10; 55% C8, 35% C10), C8 (>95% C8), C10 (>95% C10), or CO mixed 50:50 with C8-C10 or C8.

Methods: In a crossover design, 9 participants with mean ± SD ages 34 ± 12 y received two 20-mL doses of the test oils prepared as an emulsion in 250 mL lactose-free skim milk. During the control (CTL) test, participants received only the milk vehicle. The first test dose was taken with breakfast and the second was taken at noon but without lunch. Blood was sampled every 30 min over 8 h for plasma acetoacetate and β-hydroxybutyrate (β-HB) analysis.

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 as NCT 02679222. 

Brain glucose uptake is lower in Alzheimer disease (AD). This problem develops gradually before cognitive symptoms are present, continues as symptoms progress, and becomes lower than the brain glucose hypometabolism occurring in normal aging. In contrast to glucose, brain ketone uptake in AD is similar to that in cognitively healthy, age-matched controls. For ketones to be a useful energy source in glucose-deprived parts of the AD brain, the estimated mean daily plasma ketone concentration needs to be >200 μmol/L (21). With a total 1-d dose of 40 mL C8, plasma ketones peaked at 900 μmol/L and the day-long mean was 363 ± 93 μmol/L, whereas with the same amount of CO, they peaked at 300 and 107 ± 57 μmol/L, respectively. 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.

I hope Dr Pascual has read the UCLA study on bacteria mediating the effect of the ketogenic diet on seizures. I think this has big implications for how to best manage people with GLUT1 deficiency.
I can see why Nestlé are investing in C8 products to treat Alzheimer’s. It does makes sense to optimize bioavailability, but in the meantime drinking regular liquid C8 would seem a smart idea.

While C8 is being proposed for Alzheimer's as a means of compensating for reduced glucose uptake in the brain, it has other benefits.  In the next post we will look at the anti-inflammatory benefits of the ketone BHB; these benefits are very relevant to Alzheimer's, where we know that the pro-inflammatory cytokine IL-1B is over-expressed. We will discover how BHB reduces expression of IL-1B. 
The amount of C8 required to start partially fuelling the brain is trivial, just 40ml a day. If combined with BHB itself, you would need even less and if I was Nestlé that is what I would develop.
Unless you have GLUT1 deficiency I do not see why C7 is better than C8 as a brain fuel.
In autism you would only benefit from ketones as a brain fuel if you have reduced glucose uptake, reduced insulin sensitivity or a mitochondrial disorder. Clearly, some people diagnosed with autism should benefit from ketones as a secondary brain fuel to glucose. If intranasal insulin helps, ketones are particularly likely to help.



Wednesday 15 June 2016

Treating KCC2 Down-Regulation in Autism, Rett/Down Syndromes, Epilepsy and Neuronal Trauma ?

In this composite image, a human nerve cell derived from a patient with Rett syndrome shows significantly decreased levels of KCC2 compared to a control cell.  This will be equally true of about 50% people with classic autism, people with Down syndrome, many with TBI and many with epilepsy

In a recent post I highlighted an idea from the epilepsy research to treat a common phenomenon also found in much classic autism.  Neurons are in an immature state with too much intracellular chloride, the transporter that brings it in, called NKCC1, is over-expressed and the one that takes it out, KCC2, is under-expressed.  The net result is high levels of intracellular chloride and this leaves the brain in an over-excited state (GABA working in reverse) reducing cognitive function and with a reduced threshold to seizures.

The epilepsy research noted that increased BDNF is one factor that down regulates KCC2, which would have taken chloride out of the cells.  So it was suggested to block BDNF, or something closely related called trkB.

Unfortunately there is no easy way to this.  But I did some more digging and found various other ways to upregulate KCC2.

There is indeed a clever safe way that may achieve this and it is a therapy that I have already suggested for other reasons, intranasal insulin.

BDNF is a neurotrophin and other neurothrophins also have the ability to regulate KCC2. IGF-1 is another such neurotrophin and we even have very recent experimental data showing its effect on KCC2.

Regular readers will know that several trials with IGF-1, or analogs thereof, are underway.

I actually am rather biased against IGF-1 as a therapy, since in my son’s case the level of IGF-1 in blood is already high.  So I do not want to inject him with IGF-1 or even give him an oral analog.

However by using intranasal insulin the effect would be just within the CNS and insulin binds at the same receptors as IGF-1. So if IGF-1 upregulates KCC2 so will insulin.

We know from extensive existing trial data and direct feedback from one researcher that intranasal insulin is well tolerated and has no effect outside the CNS.

So rather to my surprise there seems to be a safe, cheap way to treat KCC2 down-regulation and this would also be applicable in epilepsy, traumatic brain injury (TBI) and any other condition involving immature neurons or neuronal trauma. 

The Science

There is a very thorough recent review paper that looks at all the ways that KCC2 expression is regulated.

The epilepsy researchers consider trkB, top left in the figure below.  But just next to it is IGFR which can be activated by both insulin and IGF-1.

In Rett syndrome they are already using IGF-1 to modulate KCC2.  The research is done at Penn State.

As you can see in the figure the mechanism for IGF-1 and insulin is not the same as BNDF/trkb, but Penn State have already shown that IGF-1 works in vitro.

We saw in early posts regarding intranasal insulin that this was a safe way to deliver insulin to the brain without effects in the rest of the body.

So we know it is safe and in theory it should achieve the same thing that the Penn State researchers are trying to achieve.

Signaling pathways controlling KCC2 function. The regulation of KCC2 activity is mediated by many proteins including kinases and phosphatases. It affects either the steady state protein expression at the plasma membrane or the KCC2 protein recycling. All the different pathways are explained and discussed in the main text. The schematic drawings of KCC2 as well as other membrane molecules do not reflect their oligomeric structure. GRFα2, GDNF family receptor α2; BDNF, Brain-derived neurotrophic factor; TrKB, Tropomyosin receptor kinase B; Insulin, Insulin-like growth factor 1 (IGF-1); IGFR, Insulin-like growth factor 1 receptor; mGluR1, Group I metabotropic glutamate receptor; 5-HT-2A, 5-hydroxytryptamine (5-HT) type 2A receptor; mAChR, Muscarinic acetylcholine receptor; NMDAR, N-methyl-D-aspartate receptor; mZnR, Metabotropic zinc-sensing receptor (mZnR); GPR39, G-protein-coupled receptor (GPR39); ERK-1,2, Extracellular signal-regulated kinases 1, 2; PKC, Protein kinase C; Src-TK, cytosolic Scr tyrosine kinase; WNKs1–4, with-no-lysine [K] kinase 1–4; SPAK, Ste20p-related proline/alanine-rich kinase; OSR1, oxidative stress-responsive kinase -1; Tph, Tyrosine phosphatase; PP1, protein phosphatase 1; Egr4, Early growth response transcription factor 4; USF 1/2, Upstream stimulating factor 1, 2.

The Penn State research on using IGF-1 to increase KCC2 in Rett Syndrome

The researchers also showed that treating diseased nerve cells with insulin-like growth factor 1 (IGF1) elevated the level of KCC2 and corrected the function of the GABA neurotransmitter. IGF1 is a molecule that has been shown to alleviate symptoms in a mouse model of Rett Syndrome and is the subject of an ongoing phase-2 clinical trial for the treatment of the disease in humans.
"The finding that IGF1 can rescue the impaired KCC2 level in Rett neurons is important not only because it provides an explanation for the action of IGF1," said Xin Tang, a graduate student in Chen's Lab and the first-listed author of the paper, "but also because it opens the possibility of finding more small molecules that can act on KCC2 to treat Rett syndrome and other autism spectrum disorders."

More Melatonin?

As Agnieszka pointed out in the previous post it appears that extremely high doses of melatonin can increase KCC2 in traumatic brain injury (TBI). In this example BDNF was increased by the therapy, so I think TBI may be a specific case.  In most autism BDNF starts out elevated and in epilepsy, seizures are known to increase BDNF and that process is seen as down regulating KCC2 expression.  So in much autism and epilepsy you want less BDNF.

Melatonin attenuates neuronal apoptosis through up-regulation of K+ -Cl- cotransporter KCC2 expression following traumatic brain injury in rats

Compared with the vehicle group, melatonin treatment altered the down-regulation of KCC2 expression in both mRNA and protein levels after TBI. Also, melatonin treatment increased the protein levels of brain-derived neurotrophic factor (BDNF) and phosphorylated extracellular signal-regulated kinase (p-ERK). Simultaneously, melatonin administration ameliorated cortical neuronal apoptosis, reduced brain edema, and attenuated neurological deficits after TBI. In conclusion, our findings suggested that melatonin restores KCC2 expression, inhibits neuronal apoptosis and attenuates secondary brain injury after TBI, partially through activation of BDNF/ERK pathway.

More Science

There is plenty more science on this subject.

It is suggested that in addition to IGF-1/insulin it may be necessary to involve Protein tyrosine kinase (PTK).

Protein tyrosine kinase (PTK) phosphorylation is considered a key biochemical event in numerous cellular processes, including proliferation, growth, and differentiation, and has also been implicated in synaptogenesis. Protein tyrosine kinases are subdivided into the cytosolic nonreceptor family and the transmembrane growth factor receptor family, which includes receptors for insulin and insulin-like growth factor (IGF-1). The maturation of postsynaptic inhibition may require both a cytoplasmic PTK, which increases GABAA receptor-mediated currents, and insulin, which was shown to induce a rapid translocation of GABAA receptors from intracellular compartments to the plasma membrane. KCC2 is also known to have a C-terminal PTK consensus site. Therefore, the maturation of postsynaptic inhibition may, in addition to other mechanisms, also involve the effects of PTK and insulin acting on KCC2.


I would infer from all this science that intranasal insulin is likely to increase KCC2 expression in the brain, certainly worthy of investigation.

Protein tyrosine kinase (PTK) phosphorylation is considered a key biochemical event in numerous cellular processes.  This might be a limiting factor on the effectiveness of insulin in raising KCC2.  This would then add yet more complexity.

Protein kinases are enzymes that add a phosphate(PO4) group to a protein, and can modulate its function.  A protein kinase inhibitor is a type of enzyme inhibitor that blocks the action of one or more protein kinases.

Abnormal protein tyrosine kinases (PTKs) cause many human leukaemias, so there is research into PTK inhibitors (PTK-Is).

As we know from Abha Chauhan’s mammoth book, oxidative stress controls the activities of PTK.

Monday 30 May 2016

Sense, Missense or Nonsense - Interpreting Genetic Research in Autism (TCF4, TSC2 , Shank3 and Wnt)

Some clever autism researchers pin their hopes on genetics, while some equally clever ones are not convinced.

One big problem is that genetic testing is still not very rigorous, it is fine if you know what you are looking for, like a specific single gene defect, but if it is a case of find any possible defect in any of the 700+ autism genes it can be hopeless.

Most of the single gene types of autism can be diagnosed based on known physical differences and then that specific gene can be analyzed to confirm the diagnosis.

Today’s post includes some recent examples from the research, and they highlight what is often lacking - some common sense.

There are numerous known single gene conditions that lead to a cascade of dysfunctions that can result in behaviors people associate with autism.  However in most of these single gene conditions, like Fragile X or Pitt-Hopkins, there is a wide spectrum, from mildly affected to severely affected.

There are various different ways in which a gene can be disturbed and so within a single gene condition there can be a variety of sub-dysfunctions.  A perfect example was recently forwarded to me, a study showing how a partial deletion of the Pitt Hopkins gene (TCF4) produced no physical features of the syndrome, but did unfortunately produce intellectual disability.

The study goes on to suggest that “screening for mutations in TCF4 could be considered in the investigation of NSID (non-syndromic intellectual disability)”

Partial deletion of TCF4 in three generation family with non-syndromic intellectual disability, without features of Pitt-Hopkins syndrome

This all matters because one day when therapies for Pitt Hopkins are available, they would very likely be effective on the cognitive impairment of those with undiagnosed partial-Pitt Hopkins.

Another reader sent me links to the studies showing:-

Rapamycin reverses impaired social interaction in mouse models of tuberous sclerosis complex.

Reversal of learning deficits in a Tsc2+/- mouse model of tuberous sclerosis.

But isn’t that Tuberous sclerosis (TSC) extremely rare? like Pitt Hopkins.  Is it really relevant?

Tuberous sclerosis (TSC)  is indeed a rare multisystem genetic disease that causes benign tumors to grow in the brain and on other vital organs such as the kidneys, heart, eyes, lungs, and skin. A combination of symptoms may include seizures, intellectual disability, developmental delay, behavioral problems, skin abnormalities, and lung and kidney disease. TSC is caused by a mutation of either of two genes, TSC1 and TSC2, 

About 60% of people with TSC have autism (biased to TSC2 mutations) and many have epilepsy.

How rare is TSC?  According to research between seven and 12 cases per 100,000, with more than half of these cases undetected.  

Call it 0.01%, rare indeed.

How rare is partial TSC?  What is partial TSC?  That is just my name for what happens when you have just a minor missense mutation, you have a mutation in TSC2 but have none of the characteristic traits of tuberous sclerosis, except autism.
In a recent study of children with autism 20% has a missense mutation of TSC2. 

Not so rare after all.

Mutations in tuberous sclerosis gene may be rife in autism

Mutations in TSC2, a gene typically associated with a syndrome called tuberous sclerosis, are found in many children with autism, suggests a genetic analysis presented yesterday at the 2016 International Meeting for Autism Research in Baltimore.
The findings support the theory that autism results from multiple ‘hits’ to the genome.
Tuberous sclerosis is characterized by benign tumors and skin growths called macules. Autism symptoms show up in about half of all people with tuberous sclerosis, perhaps due to abnormal wiring of neurons in the brain. Tuberous sclerosis is thought to result from mutations in either of two genes: TSC1 or TSC2.
The new analysis finds that mutations in TSC2 can also be silent, as far as symptoms of the syndrome go: Researchers found the missense mutations in 18 of 87 people with autism, none of whom have any of the characteristic traits of tuberous sclerosis.
“They had no macules, no seizure history,” says senior researcher Louisa Kalsner, assistant professor of pediatrics and neurology at the University of Connecticut School of Medicine in Farmington, who presented the results. “We were surprised.”
The researchers stumbled across the finding while searching for genetic variants that could account for signs of autism in children with no known cause of the condition. They performed genetic testing on blood samples from 87 children with autism.

Combined risk:

To see whether silent TSC2 mutations are equally prevalent in the general population, the researchers scanned data from 53,599 people in the Exome Aggregation Consortium database. They found the mutation in 10 percent of the individuals.
The researchers looked more closely at the children with autism, comparing the 18 children who have the mutation with the 69 who do not.
Children with TSC2 mutations were diagnosed about 10 months earlier than those without a mutation, suggesting the TSC2 mutations increase the severity of autism features. But in her small sample, Kalsner says, the groups show no differences in autism severity or cognitive skills. The researchers also found that 6 of the 18 children with TSC2 mutations are girls, compared with 12 of 69 children who don’t have the mutation.
TSC2 variants may combine with other genetic variants to increase the risk of autism. “We don’t think TSC is the sole cause of autism in these kids, but there’s a significant chance that it increases their risk,” Kalsner says.

"hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) is a consequence of tuberous sclerosis complex (TSC) 1/2 inactivation."

"the combination of rapamycin and resveratrol may be an effective clinical strategy for treatment of diseases with mTORC1 hyperactivation."

So for the 20% of autism with partial TSC, so-called Rapalogs and other mTOR inhibitors could be helpful, but Rapalogs all have side effects.

One interesting option that arose in my earlier post on Type 3 diabetes and intranasal insulin is Metformin. The common drug used for type 2 diabetes.


Metformin regulates mTORC1 signaling (but so does insulin).

'Metformin activates AMPK by inhibiting oxidative phosphorylation, which in turn negatively regulates mTORC1 signaling via activation of TSC2 and inhibitory phosphorylation of raptor. In parallel, metformin inhibits mTORC1 signaling by suppressing the activity of the Rag GTPases and upregulating REDD1."

Source:  Rapalogs and mTOR inhibitors as anti-aging therapeutics

Clearly you could also just use intranasal insulin.  It might be less potent but should have less side effects because it acting only within the CNS (Metfornin would be given orally).

The Shank protein and the Wnt protein family

Mutations in a gene called Shank3 occur in about 0.5 percent of people with autism.  
But what about partial Shank3 dysfunction?

Shank proteins also play a role in synapse formation and dendritic spine maturation.

Mutations in this gene are associated with autism spectrum disorder. This gene is often missing in patients with 22q13.3 deletion syndrome

Researchers at MIT have just shown, for the first time, that loss of Shank3 affects a well-known set of proteins that comprise the Wnt signaling pathway.  Without Shank3, Wnt signaling is impaired and the synapses do not fully mature.

“The finding raises the possibility of treating autism with drugs that promote Wnt signaling, if the same connection is found in humans”

I have news for MIT, people already do use drugs that promote Wnt signaling, FRAX486 and Ivermectin for example.  All without any genetic testing, most likely.

Reactivating Shank3, or just promote Wnt signaling

The study below showed that in mice, aspects of autism were reversible by reactivating the Shank3 gene.  You might expect that in humans with a partial Shank3 dysfunction you might jump forward to the Wnt signaling pathway and intervene there.

Mouse study offers promise of reversing autism symptoms

One reader of this blog finds FRAX486 very helpful and to be without harmful side effects.  FRAX 486 was recently acquired by Roche and is sitting over there on a shelf gathering dust.

Where from here?

I think we should continue to look at the single gene syndromes but realize that very many more people may be partially affected by them.

Today’s genetic testing gives many false negatives, unless people know what they are looking for; so many dysfunctions go unnoticed.

This area of science is far from mature and there may be many things undetected in the 97% of the genome that is usually ignored that affect expression of the 3% that is the exome.

So best not to expect all the answers, just yet, from genetic testing; maybe in another 50 years.

Understanding and treating multiple-hit-autism, which is the majority of all autism, will require more detailed consideration of which signaling pathways have been disturbed by these hits.  There are 700 autism genes but there a far fewer signaling pathways, so it is not a gargantuan task.  For now a few people are figuring this out at home.   Good for them.

I hope someone does trials of metformin and intranasal insulin in autism.  Intranasal insulin looks very interesting and I was surprised to see in those earlier posts is apparently without side effects.

The odd thing is that metformin is indeed being trialed in autism, but not for its effect on autism, but its possible effect in countering the obesity caused by the usual psychiatric drugs widely prescribed in the US to people with autism.

My suggestion would be to ban the use of drugs like Risperdal, Abilify, Seroquel, Zyprexa etc.

Vanderbilt enrolling children with autism in medication-related weight gain study

Here are details of the trial.

Metformin will be dispensed in a liquid suspension of 100 mg/mL. For children 6-9 years of age, metformin will be started at 250 mg at their evening meal for 1 week, followed by the addition of a 250 mg dose at breakfast for 1 week. At the Week 2 visit, if metformin is well-tolerated, the dose will be increased to 500 mg twice daily. For children from 10-17 years of age, metformin will be started at 250 mg at their evening meal for 1 week, followed by the addition of a 250 mg dose at breakfast for 1 week. At the Week 2 visit, if metformin is well-tolerated, the dose will be increased to 500 mg twice daily. At the Week 4 visit, if metformin is well-tolerated, the dose will be increased to 850 mg twice daily.