Beta hydroxybutyrate does make you smarter

Someone tweeted this study which I have read before, but unfortunately lost. I find this paper rather exceptional because of the perspective of the authors regarding congition and mental health. Most researchers only agree that a KD is efficient for seizure control, not so much for neuroprotection and neuroregeneration. The notion that the brain only functions with glucose coupled with the idea that not eating glucose is metabolically stressful (next post is about this) has deviated attention from ketone bodies as memory enhancing agents. This, I think, is one of the reasons for the very little attention given to KD for Alzheimer and Parkinson, considering the very promising results of studies done on the subject. Maybe because it is not a drug...

Back to the study. 23 older adults with mild cognitive impairment (MCI) were assigned to either a very low carbohydrate diet (VLCD) or to a high carbohydrate diet (HCD) for 6 weeks. Carbohydrate calories were 5-10% on the VLCD and 50% in the HCD. They do not controlled calories, fat or protein*. Some interesting facts from the dietary intevention:

- "The high carbohydrate diet approximated the macronutrient profile consumed at the time of enrollment for most subjects, which included at least 50% of calories from carbohydrates."
< - "We advised the high carbohydrate subjects to consume fruits and vegetables as carbohydrate sources as much as possible.Those in the low carbohydrate diet were restricted from fruit and instructed to limit carbohydrate consumption to small portions of vegetables."

Let's look at the results. 

Memory performance was only increased significantly in the low carbohydrate group. Ok, but maybe the VLCD performed better because of the changes in weight or calorie intake and not necessarily because of the diet being ketogenic. 

(I'm omitting some statistical data on the text). 

"There were significant changes in anthropometric and metabolic values and in dietary parameters. After the intervention, weight (81 kg vs. 77 kg, adjusted means) and waist circumference (95 cm vs. 90 cm, adjusted means) were reduced for the low carbohydrate group. Likewise, fasting glucose (96 mg/dL vs. 86 mg/dL, adjusted means) and fasting insulin values (14.5 /mL vs. 11.9 /mL, adjusted means) were lower for the low carbohydrate but not high carbohydrate group. Urinary ketone bodies were not detected for the high carbohydrate subjects but were present for the low carbohydrate subjects (Table 2), and ketone body levels were related to memory performance."

Table 2:

The VLCD group reduced their calorie intake considerably compared to the HCD group. Note that pre intervention energy intake was 1697 kcal for the HCD and 1762 kcal for the VLCD. There was a slight increase in protein (and fat) intake, but not sufficient to be the responsible of the sudden drop of calories. What about insulin?

"(...) semipartial correlations indicated weak and nonsignificant relationships to memory performance for each of these factors: change in calories (rsp = -0.16, p = 0.46); change in insulin (rsp =-0.26, p = 0.24); and change in weight (rsp = 0.28, p = 0.20). However, within the low carbohydrate group, the relationship between change in insulin and change in memory performance was stronger although not statistically significant, r = 0.47, p = 0.11."

Ok so there was a weak relationship between insulin levels and memory performance, but only in the VLCD. In the end, the parameter that was best correlated to memory performance was ketone body levels. 

Interesting discussion excerpts:

"A number of mechanisms might be considered with respect to our memory finding. There are indications that central ketone metabolism may confer neurocognitive benefit and mitigate neurodegenerative processes in conjunction with, but also independent of, effects on insulin. Mean fasting insulin levels prior to the intervention indicated that, on average, subjects were hyperinsulinemic. We observed a significant reduction of insulin among the low carbohydrate subjects, suggesting that the memory improvement was related, in part, to increased insulin transport into the central nervous system (CNS) as a consequence of correction of peripheral hyperinsulinemia. The trend toward a moderate relationship between fasting insulin and memory performance within the low carbohydrate group would be expected to reach statistical significance in a larger sample.It is noteworthy that a recent trial involving 12 weeks’ calorie restriction in a sample of 50 middle-aged and older adults demonstrated improvement in memory function related to change in fasting insulin (Witte et al., 2009)."

"The absence of a strong relationship between insulin reduction and memory improvement suggests that neurocognitive benefit also might be associated with other aspects of the ketotic condition. Ketone metabolism has been shown to protect hippocampal neurons from Abeta toxicity (Kashiwaya et al., 2000), glutamate toxicity, and apoptosis (Noh et al., 2006), as well as other insults such as kainic acid (Noh et al., 2003) and hypoxia (Puchowizt et al., 2005). As compared with glucose metabolism, central ketone metabolism generates lower levels of oxidative stress (Prins, 2008) and has been shown to produce greater cellular energy output and antioxidant capacity, the latter by increasing glutathione peroxidase in hippocampal cells (Veech et al., 2001; Ziegler et al.. 2003). In addition, the presence of cerebral ketones is associated with decreased apoptosis and inflammation (Gasior et al., 2006; Malouf et al., 2009), which along with oxidative stress, have been identified as fundamental factors contributing to neurodegeneration (Cotman, 2000)."

Some final notes:

- Ketone body levels were measured with urinary strips, and BOHB was not measured directly.

- The KD was by not means a high fat diet (maybe if we consider the % of calories from fat). 

* As per the authors: "Very high levels of fat (90% of total calorie intake) have been prescribed traditionally to induce ketosis for seizure management (Vining, 1998). However, recent trials have indicated that protein restriction is not necessary to achieve ketosis (Boden et al., 2005; Cassady et al., 2007) or effective seizure control (Kossoff and Dorward, 2008; Kossoff et al.,2003), allowing for a less severe regimen." More on protein intake & Ketosis here.

Krikorian R, Shidler MD, Dangelo K, Couch SC, Benoit SC, & Clegg DJ (2010). Dietary ketosis enhances memory in mild cognitive impairment. Neurobiology of aging PMID: 21130529

Extra: Glycogen, de novo lipogenesis and carbohydrates

This is an unusual post as I dont like discussing about weight loss or dietary carbs vs. fat superiority in terms of dieting. 

The metabolic advantage is a never ending discussion in the blogosphere. Recently, a more "scientific" debate has started on Peter's blog. While some prefer to argue using scientific references and basic biochemistry concepts, others use their own experience mixed with their opinions. This happens in both who defend insulin's central role in weight loss/gain and in those who state that its calories in-calories out all the way. This last group of people, when trying to dismiss the carbohydrate drives insulin drives fat storage hypothesis, use an old study as their bible. It is not surprising that the one who "popularized" this study was Carb Sane, with her famous post Nutrient fates after absorption. I wrote a quick answer some months ago, which was posted but strangely has disappeared. 

The study which basically dismisses the carbohydrate-insulin obesity hypothesis is one by Eric Jequier, called "Nutrient effects: post-absorptive interactions". I will only focus on the "Metabolic Fate of Dietary Carbohydrate" part. 

The author states:

"Although  the  glycogen  stores  are  normally maintained  within  a  relatively  narrow  range, the capacity for storing large amounts of  dietary CHO by conversion to glycogen is relatively large (Acheson et al. 1982, 1984, 1985, 1988). Fig. 1 shows that a large load of CHO (500 g dextrin-maltose  given as three meals over 5 h) to healthy subjects induces a marked stimulation of CHO oxidation over the 14 h after the first meal (240 g oxidized,  260 g stored; Acheson et al. 1985). Net lipogenesis occurred at a low rate from 5 to 10 h  after  the  first  meal, but  this lipid accumulation was  offset  by  a greater rate  of  lipid  oxidation over the next 4 h. The fat balance calculated over 14 h was negative, indicating  that the large load of  CHO did not induce a gain in  fat."

Lets review the studies used to support his statements. 

1. Glycogen synthesis versus lipogenesis after a 500 gram carbohydrate meal in man

Subjects participating in the study were 6 healthy male volunteers. They tested the metabolic effect of consuming 480g of carbohydrates from bread, jam and fruit juice, with little butter added. The results were as expected, no net lipogenesis occured, just an increase in muscle glycogen capacity. Ok, one big high carbohydrate/low fat meal in healthy subjects does not induce fat gain in the short term (10h), rather increases energy expenditure, glucose storage and oxidation. Nothing new.

2. Glycogen storage capacity and de novo lipogenesis during massive carbohydrate overfeeding in man.
Lets look at the subjects and methods used in the study:

"Three healthy young men, one of whom was a competition swimmer at university level, (21-22 y, 62-72 kg, 174-180 cm, and 11-14% body fat) with no family history of diabetes or obesity and who were not taking any medication, participated in this study."

Ok, we are talking about healthy, young and lean men. Is everyone healthy, young and lean?  I think most of the regular blog readers are people with weight/metabolic issues. The subjects included a competitive swimmer, a fact that in my opinion is very relevant, besides the fact that all of the subjects were lean. This somehow escapes from the people referencing this study. 

"The experiment lasted 14 consecutive days. During the first 3 d the subjects consumed a restricted diet, high in fat and low in carbohydrate, and followed an exercise program. Halfway through this period the subjects were admitted into a respiration chamber in which respiratory exchange measurements were to be continued for 10 d. After 36 h in the chamber the diet was changed to a high-carbohydrate, low-fat diet that was ingested for the following 7 d. During the last 2 d while still in the chamber, the subjects received limited amounts of a high protein diet (protein-sparing modified-fast [PSMF], ‘ı2.5 MJ or 600 kcal) essentially devoid of carbohydrate. The subjects then left the respiration chamber but continued to consume the high-fat, low-carbohydrate diet in restricted amounts for a further 2 d."

I wonder, if a massive carbohydrate intake does not promotes fat storage as the previous study suggested, why using a glycogen depleting protocol before an extended massive carbohydrate intake? Hmm...

In short, the protocol lasted 14 days in which:

3 first days: HFLC + exercise.

7 days: HCLF

2 days: PSMF (LF, LC)

2 last days: HFLC, restricted amounts.

So we have a scenario in which glycogen is depleted by diet plus exercise and then there is a massive high carbohydrate low fat refeed for seven days. Glycogen storage increases until the 4th day, in which glycogen stores had become saturated and de novo lipogenesis starts to increase. This shows that you need 4 days of overfeeding carbohydrates to start getting fat? No. This shows that in LEAN HEALTHY YOUNG SUBJECTS, AFTER A GLYCOGEN DEPLETING PROTOCOL, a relatively well known phenomenon called GLYCOGEN SUPERCOMPENSATION occurs. 

I dont think we can extrapolate these results to everyone nor this study shows that you can eat 500g everyday and not get fat. Specially if you are not lean and/or healthy.

3. Nutritional influences on lipogenesis and thermogenesis after a carbohydrate meal.

I find odd the use of this study to support the author's hypothesis. Subjects consumed either a high fat, mixed or high carbohydrate diet during 3-6 days preceeding the test. From the abstract (unfortunately I dont have access to the full-text):

"CHO oxidation and conversion to fat was significantly less in the high-fat diet group (222 +/- 5 g) than in the mixed (300 +/- 13 g) or high-CHO diet (331 +/- 7 g) groups, resulting in a greater glycogen storage in the high-fat (278 +/- 6 g) than in the other two groups (197 +/- 11 and 170 +/- 2 g). Net lipogenesis occurred sooner and lasted longer in the high-CHO group, amounting to 0.8 +/- 0.5, 3.4 +/- 0.6, and 9 +/- 1 g of lipid synthesized in the high-fat, mixed, and high-CHO groups, respectively. The thermic effect of the CHO load was 5.2 +/- 0.5% on the high-fat, 6.5 +/- 0.4% on the mixed diet, and 8.6 +/- 0.4% on the high-CHO diet."

This study shows what is an already known fact about high fat diets and carbohydrate loading. HFLC reduce glucose oxidation and increase non-oxidative glucose disposal, ie. glycogen stores. Although net lipogenesis was small (9g on the high carbohydrate diet), measurements were done for 24 hours. While not significant for body composition in the short term, the metabolic effect might be significant, specially considering the effect a high carbohydrate meal has on gene expression (1, 2)*. This study shows the difference between eating a 500g carbohydrate meal after a HFLC and a LFHC diet. 

4. Contribution of 500 g naturally labeled 13C dextrin maltose to total carbohydrate utilization and the effect of the antecedent diet, in man

This study is a follow up on the previous study. Nothing new to add, really. From the study:

"After the load, carbohydrate oxidation increased in each group with a corresponding decrease in fat oxidation. However, in the high carbohydrate (Fig 3C) and mixed (Fig 3B) groups, fat oxidation decreased to zero at 2.5 h and 4 h respectively after which energy expenditure was principally due to carbohydrate oxidation and de novo lipogenesis for a further 9 h and 6.5 h respectively. The negative values of lipid oxidation represent an equivalent amount of carbohydrate energy converted into lipid, ie net de novo lipogenesis. During the evening fat oxidation increased progressively and became the major energy source throughout the night in each group."

The authors concluded:

"The results imply that the more the composition of the everyday diet is rich in carbohydrate, the more full the glycogen stores become, but at the same time more carbohydrate is oxidized to provide energy. When both storage and oxidation become saturated, de novo lipogenesis can occur but not at a rate sufficient to prevent increasing blood glucose and insulin concentrations."

Summing up

It is noteworthy to mention that the author of the review, Eric Jequier participated in all of the studies shown above. The conclusion he gets from his review is the following:

"The  practical consequence  which  results from  the  different fates  of  the  ingested 

nutrients  is  that  body-weight  regulation primarily  depends on fat intake. A chronic 

excess of  fat in the everyday diet is stored and contributes to increase the adipose tissue 

mass. (...) The fat content of the habitual diet could influence the steady-state of weight maintenance. When a high dietary fat intake is chronically ingested, body-weight gain occurs."

Talk about bias. Although he mentions that "The practical implication of these findings is  that the proportion of fat and CHO energy plays an important role in body-weight regulation" he goes on saying that "The ad  lib. consumption of high-CHO-low-fat  diets induces a slow rate of  weight loss, with a spontaneous decrease in energy intake" and "Prevention of obesity should benefit from this recent advance in our knowledge of the post-absorptive effects of nutrients".

I did a quick search on more studies done by Jequier and I found these gems:

Low fat diets are preferred

Is fat intake a risk factor for fat gain in children

Failure of dietary fat intake to promote fat oxidation: a factor favoring the development of obesity

Pathways to obesity

I encourage you to check the references used in the section "Metabolic Fate of Dietary Fat". 

Studies can show whatever the author wants to. The only way of knowing and interpreting the real data is reading the data source for yourself. Or reading trustful blogs. But hey, after all, fat is what makes us ugly, fat and ultimately kills us, doesnt it?

* Some further reading on ChREBP is recommended.

Jéquier E (1995). Nutrient effects: post-absorptive interactions. The Proceedings of the Nutrition Society, 54 (1), 253-65 PMID: 7568258

Ketomyths II

In the first post of these series I reviewed the evidence regarding mucin deficiency and glucose restriction. Its time to move on into a more complex topic: vitamin C and scurvy. The best article written on low carbohydrate diets and scurvy comes again from Dr. Jaminet. I will use some of his statements to explain my points of view on the subject. Before I start, I have to mention that ketogenic diets for epilepsy are not comparable to a paleoish ketogenic diet. It is too high in fat, too low in protein and the main source of fat are heart healthy vegetable oils high in n-6 PUFA. Most of the time patients are also water-restricted to increase blood ketone levels. Having said this, I will not discuss any example which uses an anti-epileptic ketogenic diet in the article (except the one on selenium deficiency, which is relevant for the post).

First, it is important to understand that ascorbate is asborbed in the small intestine modulated by glucose (1):

Na+ dependent glucose transport via SGLT1 receptors in enterocytes modulates the asborption of ascorbate, while transport of dehydroascorbic acid (DHA) is by facilitated difussion. As glucose content in the lumen and in the enterocyte increases, ascorbate transport is reduced. In layman's terms: ascorbate absorption is reduced by high glucose, so if you eat more glucose you will need to eat more ascorbate. This means, glucose increases your minimal requirement for vitamin C in your diet. 

But once ascorbate and DHA enter the bloodstream, they must be transported inside cells. 

"DHAA can be recycled back into vitamin C, but only inside cells. In order to enter cells, DHAA needs to be transported by glucose transporters. GLUT1, GLUT3, and GLUT4 are the three human DHAA transporters; GLUT1 does most of the work."

 Structurally, DHAA and ascorbic acid are similar to glucose:

Ascorbic acid, reduced form (left) and DHA, oxidized form (right)

This could be one of the reasons for the sharing of membrane transporters. The affinity of GLUT4 for DHA is low, so the main transporters are GLUT1 and GLUT3 (2), that is, glucose and DHA are both competitive substrates for some glucose receptors. High glycemia reduces the transport of DHA into cells. 

Jaminet states:

"Glucose transporters are activated by insulin. Thus, DHAA import into cells is increased by insulin, leading to more effective recycling of vitamin C [8]"

The reference takes us to a study done on osteoblastic cells (3). When these cells were incubated with insulin, the uptake of DHA and the intracellular concentration of ascorbate from DHA increased. But if you read look into the results, you will find: 

"Transport of [¹⁴C]DHAA was inhibited by D-glucose and 2-deoxyglucose, both of which are substrates of GLUT"

What? But isnt insulin, which is stimulated by glucose, supposed to increase DHA transport? Yes, but here is where interpretation problems arise. When we study cells in vitro we isolate the variable we want to study. This helps us understand the exact mechanism of action of certain molecule or pathway (because there is no other molecule/pathway influencing it) but this also creates a problem: our cells are not isolated. In fact, they are under many different convergent signals which might have sinergistic or antagonistic effects on one cellular process. If we focus only on the proximal cause (insulin increases DHA uptake) we miss the big picture for understanding metabolic processes (why insulin increases DHA uptake?). From my point of view, this is an evolutionary acquired mechanism. Vitamin C transport/recycling modulated by insulin/glucose can be illustrated using a different scenario: muscle protein balance. Insulin's role in different tissues is mainly inhibitory. In skeletal muscle, insulin inhibits muscle protein breakdown. But has a passive role on muscle protein synthesis, which is stimulated by plasma amino acid availability. On the other hand, the most powerful stimulus for insulin secretion, hyperglycemia, increases proteolysis (4). Hence, insulin counteracts the effect of hyperglycemia (until a threshold is reached) on muscle protein balance. This effect is also illustrated by glucose induced inflammation. Hyperglycemia is inflammatory (5, 6, 7) and contrary to popular beliefs, insulin is anti-inflammatory (8, 9). My guess is that glucose is the main stimulus for insulin secretion because it is inflammatory. Just like in skeletal muscle, insulin is released to prevent damage from glucose. 

Getting back to the topic, these examples help to understand why insulin might increase DHA transport and recycling. When there is a higher level of glucose competing with DHA, insulin's role is to increase the number of GLUT receptors for achieveing a normal intracelular DHA level for proper conversion to ascrobic acid (AA). 

There are other vitamin C transport systems used by cells, namely SVCT1 and SVCT2, two sodium dependent transporters for AA, which are regulated among others, by AA plasma concentrations. This transport is not regulated by insulin. So we have two ways by which vitamin C is transported into cells (10):

AA is transported inside cells by Na+ dependent transporters coupled to a Na+/K+ATPase, while DHA is transported through GLUT proteins. DHA is rapidly reduced to AA, which can excit the cell by different uncharacterized mechanisms (beyond the scope of this post).

Remember that DHA is the oxidized form of AA, which explains its antioxidant nature. AA is oxidized to the ascorbyl free radical (transfering of one electron to a metabolic oxidant) which is further oxidized, losing a second electron, producing finally DHA (this is illustrated in the second figure in the post, although simplified and the ascorbyl free radical is not shown). If there are more oxidants, higher levels of AA are needed to reduce them so there is an increase in the DHA/AA plasma ratio, as observed in diabetics. Regarding this issue, Jaminet says:

"Confirming the role of insulin in promoting vitamin C recycling, Type I diabetics (who lack insulin) have lower blood levels of vitamin C, higher blood levels of DHAA, increased urinary loss of vitamin C metabolites, and greater need for dietary vitamin C. [9, 10]"

The first mistake is comparing no insulin (such as in type I diabetics) with low insulin. But why do type I diabetics have lower levels of AA and higher levels of DHA? Is it because a defective recycling via lower DHA cell uptake? Maybe. But I dont think it is the only cause. Hyperglycemia, as mentioned earlier, produces oxidative stress, increasing the formation of reactive oxygen species which react with AA, oxidizing it to form ultimately DHA. Because there is a defective transport of DHA into cells (because of competitive inhibition by glucose and abscence of insulin), the balance is shifted towards DHA. The main source of mitochondrial ROS seems to be complex III during the oxidation of complex I substrates (NADH dehydrogenase) (11). Without going into details (maybe on a different post), complete (aerobic) glucose oxidation produces a ratio of NADH to FADH of 5:1. This means that mitochondrial energy metabolism relies more on complex I than complex II, increasing the production of ROS. This scenario is also observed in type II diabetics (12, 13), who in fact have the opposite (chronic hyperinsulinemia), but glucoregulatory alterations. Despite having high insulin levels, they have low plasma vitamin C. Increased ROS production both by systemic inflammation, hyperglycemia, carbohydrate-based mitochondrial energy production and defective cellular transport are the likely causes of vitamin C alterations observed in these patients. In uncontrolled TIDM, abscence of insulin makes things worse. This is by no means applicable to ketogenic diets.

"Dehydroascorbate, the fully oxidized form of vitamin C, is reduced spontaneously by glutathione, as well as enzymatically in reactions using glutathione or NADPH. [11]"

Then, Jaminet states: 

"Glutathione is recycled by the enzyme glutathione peroxidase, a selenium-containing enzyme whose abundance is sensitive to selenium status. One difficulty with zero-carb diets is that they seem to deplete selenium levels."

For this assupmtion, he references a study on a sudden cardiac death on a ketogenic diet for seizure control (14). From the cited study:

"Selenium is an essential nutrient in the human diet. Sources of selenium include cereals, meats, and fish [8]. However, depending on the intake of those foods and the source of cereals (e.g., soil rich or poor in selenium), selenium levels in humans are variable. Patients on the ketogenic diet have little cereal intake, and only moderate fish and meat intake, and thus are predisposed to low selenium levels."

Even I said I wasn't going to refer to seizure-control ketogenic diets, its worth mentioning that according to the last recommendations of the International Ketogenic Diet Study Group (15), the "normal" diet used in this cases is 90% fat and 10% of carbohydrates and protein combined. They state that "Calories are typically restricted to 80%–90% of the daily recommendations for age". Not very similar to popular paleo-keto diets. To achieve this incredibly low level of protein, they have to restrict important sources of Se, specially those eaten in abundance by low carbers like meat and fish, being the former one of the foods with best Se bioavailability (16). The recommended Se intake seems to be around 40ug/day (17) and muscle meats, on average, have 0.3-0.4mg Se/kg (organ meats such as liver and kidney concentrate more Se, from 4 to 16 fold the amount on muscle). Remember that 1000ug = 1mg, so 0.4mg = 400ug. If you eat 100g of meat a day (excluding other animal sources) you are eating 40ug of Se, the recommended intake (for reference, 100g equals to 3.5oz/0.22lb).  

Jaminet concludes:

"So here we have a second mechanism contributing to the development of scurvy on a zero-carb diet. The diet produces a selenium deficiency, which produces a glutathione deficiency, which prevents DHAA from being recycled into vitamin C, which leads to DHAA degradation and permanent loss of vitamin C."

As we have seen, using a clinical case based on a ketogenic diet for seizure control leads to a flawed conclusion. 

Finally, as said by one of the PHD readers in the comment section, ketogenic diets have shown to increase GSH levels (18).

The hypothesis proposed by Jaminet is not supported by facts, only by assumptions based on misinterpretation of the existing data. Proper ketogenic diets dont produce vitamin C, glutathione or selenium deficiency. Loss of glucoregulation does.

Quick on starch, ketosis and non-oxidative glucose disposal

Post-exercise ketosis is a phenomenon rarely discussed these days. But there is some considerable number of research done on this subject. A very interesting study done in the 80's tested the effects of alanine, glucose and starch ingestion on starvation ketosis and post-exercise ketosis (1).

"The effect on the blood ketone body concentration of a 100 g oral dose of either alanine, glucose or starch was studied in forty-four healthy men. Twenty of the subjects were highly trained long-distance runners who underwent 'glycogen stripping' as previously described by us (Koeslag et al. 1980). Twelve non-athletic subjects fasted for 65 h before the test, and twelve were studied on a normal day after a normal breakfast."

Trained athletes were tested after running 2 hours. They ate a low carbohydrate diet for 48h prior to the experiment. The starvation group was composed of 12 non trained subjects, fasted for 65 hours. 

Results

The arrow shows the point in which the different solutions were ingested.

"The ingestion of glucose or alanine after exercise caused the mean blood ketone body concentration to fall to less than 0 5 mmol/l in 2 h. The fall was more prompt, less variable and longer lasting after 100 g alanine was ingested than after 100 g glucose. At 15.00 the mean blood ketone body concentration was rising again in the subjects who had taken glucose, but not in those who had taken alanine. The difference between the mean blood ketone body concentrations of the two groups at 15.00 is statistically significant (P < 0-01).

(...) Starch ingestion caused the blood ketone body concentrations to fall, but to a lesser extent than after alanine or glucose ingestion. After starch ingestion, as after glucose, the mean blood ketone body concentration was rising again at 16.00 (Fig. 1), thus reaffirming the evanescence ofthe antiketogenic effects of carbohydrate administration."

So the antiketogenic effect was alanine > glucose > starch. We are talking about 100g of each, after either 65h of fasting (almost 3 days) and running 22km plus a low carbohydrate diet. This is by no means a "typical" scenario. But it shows us that starch is less antiketogenic than glucose. Evolutionary reasons perhaps? 

Extrapolating the findings to real life situations, there shouldnt be much of a problem about getting back to ketosis consuming carbohydrates only post workout, specially if you follow a very low carbohydrate diet, fast daily and train in a fasted state. This is the winning combo for minimizing glucose oxidation and maximizing non-oxidative glucose disposal after a glucose load. 

Studies have shown that adaptation to a high fat-low carbohydrate diet produces a shift in glucose metabolism, reducing glucose oxidation and increasing glycogen synthesis and glucose storage. A similar metabolic response is triggered by short term fasting and resistance training. 

Further reading on high fat diets and glucose metabolism:

Dietary fat content alters insulin-mediated glucose metabolism in healthy men.

High-fat/low-carbohydrate diet reduces insulin-stimulated carbohydrate oxidation but stimulates nonoxidative glucose disposal in humans: An important role for skeletal muscle pyruvate dehydrogenase kinase 4.

Low-carbohydrate diet alters intracellular glucose metabolism but not overall glucose disposal in exercise-trained subjects.

Carbohydrate refeeding after a high-fat diet rapidly reverses the adaptive increase in human skeletal muscle PDH kinase activity.

Koeslag JH, Noakes TD, & Sloan AW (1982). The effects of alanine, glucose and starch ingestion on the ketosis produced by exercise and by starvation. The Journal of physiology, 325, 363-76 PMID: 7050344

Ketomyths

I recently started a discussion with Dr. Harris, author of PaNu. If you have read my posts, you will see that I refer to his website many times because I share almost all of his ideas. But one critical topic in which we differ is ketosis. I find it very interesting, considering that Peter also avoids ketosis permanently, and both of them are the two bloggers who I respect the most.

The answer posted by Dr. Harris lead me to find many ketomyths in the "paleosphere" and "blogosphere". Although most don't think ketosis is dangerous, the common perception is that "more is not better", that is, being in ketosis 24/7 is not better than being only for short periods of time. According to Harris, there is no need to if you aren't sick. The main argument is that ketosis is not the natural state of the body and represents a metabolic stress. Keep in mind I never said that we should be in ketosis 24/7, I just said that ketosis should be the baseline physiological state (so most of the time you are in, but not necessarily 24/7*). 

Initially, I was going to address the points made by Dr. Harris in his post but I found one ketomyth that has spread all over the internet which people use as an argument against glucose deprivation. Before I go on I must say that zero carb diets are only theoretically possible, unless you eat only yolks and oil. 

It all started with the incidence of gastrointestinal cancers in long time OD's that were reported on Peter's blog. People started speculating on the origin of this issue trying to understand why the "Optimal" diet was not that optimal. The Jaminet's, authors of the Perfect Health Diet, posted an article attributing mucin deficiency as the cause. I think this can be a likely cause but only in OD dieters. The OD is characterized by restricting both protein and glucose. But somehow people misinterpreted this and started using it as an argument against all ketogenic diets. 

But what exactly determines GI mucin production? Mucin biosynthesis is highly sensitive to protein malnutrition, specially threonine deficiency (1,2,3). Other aminoacids like serine, proline and cysteine can also promote mucin synthesis (4). When intestinal epithelial goblet cells are deprived of glucose, butyrate modulates MUC gene expression and becomes the main regulator of mucin synthesis (5). There is no evidence that the abscence of dietary glucose affects mucin synthesis. So any deficiency of mucin in ODs would occur because of a low protein intake, not because of a glucose deficiency (call it a secondary-glucose deficiency if you wish). 

Thr, Ser, Pro and Cys are all glucogenic aminoacids. The demand for these amino acids increases when eating a very low carbohydrate diet because gluconeogenesis is increased to maintain a normal blood glucose level. Eating a diet low in these amino acids could compromise mucin production because they are redirected to glucose production instead of other biosynthetic pathways. 

Dr. Jaminet goes on and explain:

Throughout my 2 years on this zero-carb diet, I had dry eyes and dry mouth. My eyes were bloodshot and irritated, and I had to give up wearing contact lenses. Through repeated experiments, I established that two factors contributed to the dry eyes – vitamin C deficiency and glucose deficiency. After I solved the vitamin C issue, I did perhaps 50 experiments over the following few years, increasing carbs which made the dry eyes go away and reducing them which made them immediately come back. This established unequivocally that it was a glucose deficiency alone that caused the dry eyes.

I find this last line specially interesting, considering that bOHB has shown to be protective in different types of dry eye models (6, 7), although used as eye drop. 

What about Vitamin C? Thats the topic for the next post. 

I am not trying to put down Dr. Jaminet's work. On the contrary, I find it highly valuable, specially the emphasis he has on micronutrition. I also like n=1 experiments. But I think glucose deficiency is a misnomer. Think about protein deficiency and dehydration instead. These problems should not arise if you eat a proper amount of protein, drink enough water during the day and eat a fairly good amount of sodium (is salt paleo?) and potassium. 

Beta hydroxybutyrate might make you smarter

During physiological ketosis, KB can supply almost 60% of the brain's energy requirement. Plasma KB pass the BBB through monocarboxylic acid transporters (MCT) in a gradient-dependent manner and are readily available for neurons and astrocytes (which are also ketogenic (1)). In fact, the brain is happy without glucose and using ketones (2). Besides being neuroprotective and metabolically more efficient, the main KB, bOHB (3-HB), could enhance memory and learning.

Zou et al (3) tested this hypothesis in mice:

"This study sought to investigate the effect of 3-HB and derivatives on neuroglial cell metabolic activity and gap junctional intercellular communication of hippocampal neurons, to evaluate the hippocampal expression of PUMA-G and proteins related to memory following treatment with 3-HB, and to determine whether 3-hydroxybutyrate methyl ester (3-HBME) improves learning and memory in the normal mouse."

Interneuronal communication can occur indirectly and directly. In the former, chemical synapses are involved, in which transmitters are released into the extracellular space and bind the postsynaptic cell membrane. In the latter, electrical synapses mediate communication. The most prevalent group of electrical synapses, neuronal gap junctions, connect directly the intracellular space of two cells by gap junction channels. Connexin proteins are the structural components of gap junction channels in the nervous system. Specifically, connexin 36 has been involved in learning and memory (4).

3-HB is the endogenous ligand for PUMA-G (5). Its activation in adipose tissue is also produced by nicotinic acid, inhibiting lipolysis and controlling the rate of ketogenesis. This represents an homeostatic mechanism by which 3-HB controls its own production, preventing ketoacidosis. 

Researchers used the Morris water maze for measuring learning and memory, and analyzed hippocampal neuron exposure to 3-HB and derivatives in vitro. All of the metabolites stimulated metabolic activity in neuroglial cells. 3-HMBE increased gap junction intercellular communication, as well as connexin 36 expression by 30% (compared to 12% in mice treated with acetyl-L-carnitine) and pERK2 levels (phosphorylated ERK2, necessary for connexin assembly). Moreover, PUMA-G mRNA was found in the hippocampus, subthalamic nucleus, temporal cortex and frontal cortex, and 3-HBME enhanced the transcription of PUMA-G in the hippocampus.

So this translates to improved memory and learning?

"The escape latency of mice in all groups decreased with time. Overall treatment comparisons indicated that statistically significant differences existed among the groups. Notably, the 30 mM 3-HBME groups took less time (p < 0.05) than the control groups on days 1 and 3–5 to find the platform (Fig. 5B). All treatment groups were faster (p <0.05) than the control group on day 5."

Treatment groups include mice treated with either 3-HBME (20, 30 or 40mg/kg/d) or acetyl-L-carnitine. Control group was water. 

"Paths taken to the platform area on the fifth day of spatial training by mice in the 30 mg/kg/d 3-HBME group were more direct than those taken by mice in the control group, which took more circuitous paths."

Ketotic mice knew exactly what they wanted. 

This causes less total swimming distance (ie. increased efficiency):

"Similar to the escape latency results, the total swimming distance of the 30 mM 3-HBME groups was shorter (p <0.05) than the control groups on days 1 and 3–5. (...) Moreover, the total swimming distance of mice in the 30 mg/kg/d 3-HBME group was shorter (p <0.05) than that of the control or other treatment groups at days 3–5."

A probe test* then was performed to evaluate memory. Three parameteres were measured: the number of crossings of the exact place where the platform had been located, the swimming distance in the quadrant of the former platform position, and the swimming path in the pool. 

Number of times that mice crossed the former position of the hidden platform within 60s:

 

Swimming distance in the platform quadrant:

Swimming path:

Finally, thigmotaxis was lowest in the 30mg/kg/d 3-HMBE group.

A retention test was performed two days after the probe test: 

These test showed that the 30mg/kg/d 3-HMBE group:

a. Crossed more times the exact place where the platform had been located,
b. Had the larger swimming distance in such quadrant,
c. Found the platform faster than the other groups, 
d. Were the most calmed (less thigmotaxis), and
e. Found the platform faster than the other groups during the retention test

These results suggest that 3-HMBE, given at 30mg/kg/d, enhanced learning and memory**.

Researchers used polyhydroxybutyrate (PHB) (a polyhydroxyalkanoate) to produce 3-HB and derivatives. Can we extrapolate these results to physiologically produced 3-HB? The finding that hippocampal neurons expressed PUMA-G receptors is encouraging. As mentioned, 3-HB is the endogenous ligand for PUMA-G, but there seems to be a desensitization of the receptor when exposed to large amounts of 3-HB compared to 3-HBME and 3-HBEE. As PUMA-G interaction with several ligands is not well understood, we cannot draw many conclusions. 

Some studies have evaluated the potential benefit of 3-HB in neurological disorders which compromise memory and learning, such as Alzheimer's disease (AD). For example, in some patients with AD and mild cognitive impairment, MCT oil produces an improvement on cognitive testing and paragraph recall (6), correlated with the increase in plasma ketones. This neuroprotective effect has been explained by the increase in metabolic efficiency associated with 3-HB (7) and inhibition of apoptosis (8), but it might also act through PUMA-G and connexin 36 dependent mechanisms. Further studies should help discovering the molecular pathways involved. In the meantime, maybe schools should start giving students coconut oil shots instead of skim milk.

* The platform was removed from the pool and mice were challenged to a single search trial for 60s. 
** Note that this group performed best than 20mg/kg/d and 40mg/kg/d.


Zou XH, Li HM, Wang S, Leski M, Yao YC, Yang XD, Huang QJ, & Chen GQ (2009). The effect of 3-hydroxybutyrate methyl ester on learning and memory in mice. Biomaterials, 30 (8), 1532-41 PMID: 19111894

Integrative Metabolism & Physiology: The case for Lipotoxicity

When trying to understand human metabolism and physiology, one must consider an organism as a whole. Because of our human nature, we tend to synthesize most of the available information to the point of applying the Occam's razor principle incorrectly. Living organisms are open systems in which every metabolic pathway is interrelated, maintaining a dynamic steady state. This is one of the main issues of studying cells in vitro versus in vivo.

As I pointed out in my introductory post, every human biological process has its purpose. There are not "bad" molecules or "good" ones. Glycolysis is not bad, excessive glycolisis is bad. Lipolysis is not bad, excessive lipolysis is bad. And so on. Considering this principle is essential for understanding modern diseases, treating and preventing them. Modern medicine has made the huge mistake of applying the bad/good concept for trying to understand diseases. We should not try to understand diseases only by proximate causes, but by evolutionary causes as well (for an in depth review, see Harris and Malyango, 2005). 

Lately, IR is one hot topic in the scientific community as well as in the blogosphere. Because of its implication in almost every modern disease, many apply the "morality principle", by which IR is bad and IS is good. If you ask the regular health reader the question "Is IR bad?" you will probably hear an unanimous YES!. On the contrary, my answer would be "it depends". 

To undestand why something completely normal like IR goes pathological we have to look at the Randle Cycle. In a nutshell, every substrate promotes its own oxidation. If you eat more glucose, you burn more glucose; if you eat more fat, you burn more fat*. The human body has adapted to use both sources of fuel as energy. Early humans evolved in highly different ecological niches, some with an increased amount of sugar/starch and others with less or none. Mechanisms should have been developed for coupling both energy substrates. When the main source of fuel is glucose, the expression of key enzymes involved in glycolisis increases. Glut-4 translocation increases for clearing glucose more efficiently from the bloodstream. Insulin reduces the rate of lipolysis by downregulating HSL, so less FFA are used for energy. Glucose itself promotes its storage, both as glycogen or fat, directly or indirectly (activation of hepatic/adipose DNL, ChREBP and several lipogenic genes, etc.). On the contrary, when you eat more fat, you will use more fat. Fat ingestion produces an increase in lipolysis and beta-oxidation. CPTI, UCP, CD36/FAT are all increased. Because the insulin response to fat is null, HSL is not supressed and lipolysis serves to deliver the necessary energy for the tissues. If more FFA than needed are released, re-esterification occurs. Excess flux of FFA to the liver produces KB to reduce the need for glucose and to control the rate of lipolysis. 

There are some cells that can only use glucose. GnG is the mechanism by which we evolved to supply this demand in a coordinate way: there is never going to be "too much" glucose produced by GnG. When carbohydrate intake is reduced drastically, we supply the exact amount to these cells by this mechanism. We rely less on glycogenolisis and more on GnG for controlling glycemia. Peripheral IR develops to redistribute the produced glucose to the glucose-strict using cells. The muscle functions as well or better with FFA and KB, and has its own glucose reserovir. There is no evolutionary logic on relying on glucose when you have more efficient fuels readily avilable. Palmitate is the key metabolic mediator in this process. It serves as an intercellular signal that integrates energy metabolism, reducing the utilization of glucose and increasing fat oxidation. Peter from Hyperlipid has written about this before in his Physiological Insulin Resistance series.

In a normal physiological scenario the body is adapted to handle increased amounts of glucose or FFA in plasma. Is abnormal to have both substrates high at the same time. If this happens it means that you have a dysregulated metabolism. And here is where the main problems of interpretation arise when evaluating pathological IR. Some say the culprit is high glucose. Others say its lipotoxicity. This last mechanism has gotten much attention lately because of the rise of carb conscious bloggers who dismiss the insulin/carbohydrate hypothesis. Lipotoxicity is the mechanism by which high plasma FFA concentrations produce deleterious metabolic effects. Lets think for a second. We store energy as fat. We use energy as fat. We store fat mainly as palmitate (the principal muscle IR agent and responsible for modern diseases). When we need energy, we hydrolize TG and free palmitate into plasma. High palmitate and high FFA produces lipotoxicity and IR, so then, are we designed to kill ourselves? The answer is obviously no. Lipotoxicity only occurs if there is a mismatch between lipolysis and beta-oxidation. For instance, there is evidence that saturated FA trigger a specific inflammatory response in coronary artery endothelial cells (1), lipoapoptosis (2) and endothelial dysfunction (3). When skeletal muscle cells are exposed to increased levels of palmitate, we see that the deleterious effects build-up dose dependently (4). This means that while myocites can handle a physiological increase in palmitate, they start to develop defense mechanisms when levels rise to pathological. This only occurs in abnormal or broken** metabolisms, like in T2DM. Having endothelial cells chronically exposed to very high FFA is bad, so muscle cells try to reduce this exposure by storing lipids as IMTG. Accordingly, IR has been proposed as a defense mechanism for controlling body fat distribution (5). NEFAs have also shown to impair insulin secretion, possibly as a preventive mechanism (6).

So for lipotoxicity to occur, there must be a metabolic dysregulation by which the rate of lipolysis is not controlled. As we all know, insulin is the key enzyme controlling HSL. It has been proposed that adipose tissue IR (ATIR) is the starting point in the pathogenesis of pathological IR (7). Without insulin, lipolysis is unregulated. Because this is not due to a physiological need***, oxidation is not correlated with lipolysis and FFA start to rise in plasma and accumulate in extra-adipose tissues. But pathological IR is not characterized only by  ATIR. Loss of control of GnG also occurs because of hepatic IR, producing hyperglycemia. Now the body has two potential fuels in excess and each one promotes its own oxidation. To compensate, hyperinsulinemia occurs and aggravates the situation. Chronic hyperinsulinemia and hyperglicemia, without an excess of FFA, have shown to impair insulin sensitivity and insulin secretion (8) by a different mechanism (impaired non-oxidative glucose disposal). This, combined with high plasma FFA is a recipe for disaster. 

When talking about lipotoxicity one must be careful with the evidence. As I stated, we cannot make conclusions based only on in vitro studies. When you eat no carbohydrates (or at least not intentionally) FFA will and should rise in plasma. Its completely normal, you use fat as fuel, you need it available. Just like ketosis. But if you eat carbohydrates, you shouldn't have elevated FFA nor ketones. This is when lipotoxicity occurs. 

Bottomline: Each metabolic fuel controls its own oxidation. We cannot use isolated mechanisms or variables to try to understand physiology and metabolism as the body is a highly regulated and integrated system. Arguing that FFA in plasma cause lipotoxicity is as misleading and wrong as saying that insulin by itself causes insulin resistance. 

* Not necessarily your own body fat. 
** As per the definition of Dr. Harris.
*** You need energy = you burn more fat (increased rate of lipolysis). FFA are supplied according to the energy demand and oxidized.