Safe starches, blood glucose and insulin

A reader asked me recently about a subject which is confusing many people in the paleosphere. 

"Jaminet in his debate with Rosedale suggests higher carb diets tend to lower blood sugar whereas low carb diets, RAISE it. Can you help me untangle what is going on here? It makes it sound like the more carbs you eat the better your blood sugar levels which does not seem right to me. Clearly, an important health goal is achieving low blood glucose so I would want to know what is the best way to eat to control them. 

I always assumed that the less sugar you eat, the less blood sugar you'll have. Is there a threshold? If Jaminet is correct, then shouldn't we see a higher fasting glucose associated with ketogenic diets that are totally carb restricted vs higher carb diets? I know excess protein might be converted into glucose but if you followed a ketogenic diet with low protein would you still see the rise in blood glucose? 

What is the mechanism through which blood glucose is being lowered in high carbers? Do they secrete more insulin to deal with it hence lower blood glucose? Would their insulin levels therefore be higher even if blood glucose was relatively low? Which is worse for health- low glucose/high insulin or moderate glucose/low insulin? 

What is the mechanism through which some cancers are being suppressed with ketogenic diets if not through lowered blood glucose? Is there something else going on?"

These are very important questions as they raise some concern in people which utilize a low carbohydrate diet for controlling their blood glucose (BG). Before trying to elaborate an answer, there are some facts that must be kept in mind:

• Hyperglycemia is not a disease, it is a symptom. 
• BG levels can be affected by non-dietary factors.
• The two principal energy substrates for humans (glucose and free fatty acids (FFA)) compete with each other for their utilization.
• Calories matter.
• There are differences between physiological insulin resistance (PIR) and pathological insulin resistance (PaIR). The term "insulin resistance" is very vague, it doesn't define explicitly which tissue(s) is IR.

The first issue to adress is whether ketogenic diets raise BG levels. The only evidence I have seen for this happening is in anecdotes from people in the internet. But if we want to have an objective look at the subect, we must see what happens in studies done with ketogenic diets (I will use low carbohydrate and ketogenic diets equally). 

Ketogenic diets and blood glucose levels

Most studies done on ketogenic diets are short-term and involve weight loss. All of them show a reduction in BG and insulin levels. A study by Grieb et al. (1) found that people eating an optimal diet (Kwasniewski) had on average a BG level of 87.9mg/dL, which is in the normal range; and very low values of HOMA-IR. Sharman et al. (2) have shown that the metabolic benefits of carbohydrate restriction are independent of weight loss. Given the evidence, it is only possible to speculate about the mechanisms by which BG levels rise in some people eating a ketogenic diet.

The goal of a ketogenic diet is to simulate fasting, but without the negative effects of prolonged nutrient restriction. Before going on, it is pertinent to remember the Randle Cycle (3):

In short, both FFA and glucose compete with each other for their uptake and oxidation. This can be translated as when BG is high, FFA utilization is low; and when BG is low, FFA utilization is high. Ketogenic diets are characterized by low BG levels, in part because of a drastic reduction in exongeous glucose. In turn, plasma FFA rise from dietary and endogenous sources (the contribution of each one depends on energy balance). During this scenario, plasma ketone bodies also rise. So we have the following metabolic milieu:

Cellular effects of a fasting-type metabolism

For exerting it metabolic effects, insulin needs to first bind its receptor (the insulin receptor, IR). Upon binding, insulin triggers an intracellular signaling cascade, which influences both the function of intracellular proteins and gene expression. The signaling pathway triggered by the binding of insulin include the recruitment of the IRS (insulin receptor substrate) to the cytosolic part of the insulin receptor dimer. The signaling cascade stimulated by insulin is essential for its function. If proteins involved in this signaling pathway are inhibited, there will be no cellular response to the binding of insulin and the activation of IR.

One level of inhibition of glucose utilization by FFA involves the inhibition of GLUT4 translocation to the plasma membrane. The translocation of GLUT4 is stimulated by insulin, by activation of IRS, PI3K and other proteins (4, 5). In vitro studies have shown that palmitate, the main fatty acid stored in mammalian adipose tissue, inhibits GLUT4 translocation and activity (6, 7). This results in reduced glucose uptake in skeletal muscle.

Inactivation of PDH (pyruvate dehydrogenase) is one of the most important mechanisms for inhibition of glucose oxidation  by FFA. PDH activity is controlled by phosphorylation, by PDK (pyruvate dehydrogenase kinase) and PDP (pyruvate dehydrogenase phosphatase). Phosphorylation by PDK inactivates PDH, while dephosphorylation by PDP activates it. Fatty acid oxidation increases the mitochondrial ratios of [acetyl-CoA]/[CoA] and [NADH]/[NAD+], which inhibit PDH. Low carbohydrate diets have shown to reduce muscle PDH and increase PDK (8, 9), effects which are reversed by a carbohydrate refeeding (10). Fatty acids also increase the concentration of cytosolic citrate, which inhibits 6-phosphofructo-1-kinase, providing another mechanism of inhibition of glucose oxidation (3). Fatty acids can reduce phosphorylation of IRS-1 (11), GSK-3b and PKB/Akt (12), thereby acting also downstream of IR and IRS.

The metabolic response to fasting can show what are the effects on insulin signaling of very high levels of plasma FFA and ketone bodies, but within a physiological range. In a very interesting study, Soeters et al (13) found that insulin-mediated peripheral glucose uptake after 62h of fasting was significantly lower compared to 14h of fasting. They also found that after 62h of fasting, Akt phosphorylation at Ser473 and AS160 phosphorylation at Thr642 were reduced. This implies that insulin signaling was attenuated (reduced phosphorylation of Akt) as well as glucose uptake (phosphorylation of AS160 is involved in the translocation of GLUT4). The authors concluded:

"(...) it is possible that pAKT-ser⁴⁷³ is involved in the physiological adaptation to fasting, inducing a reduction in peripheral glucose uptake and protecting the body from hypoglycemia."

Intramyocellular triglyceride accumulation is thought to mediate fatty acid insulin resistance. This is one way by which some authors think that a high-fat diet leads to insulin resistance. Compared to fasting (67h) a very low carbohydrate diet (eucaloric) produces the same amount of IMTG accumulation, both produce glucose intolerance and reductions in insulin sensitivity (14). Thus, the factor for triggering this metabolic response seems to be the absence (or drastic reduction) in glucose availability (my bolds):

"Thus, we suggest that dietary-induced IMTG accumulation and insulin resistance in healthy humans may be largely influenced by circulating FFAs, whose availability (in turn) is regulated by dietary CHO intake. (...) our study provides support for the hypothesis that the physiological trigger for this coupling in the healthy individual may be a short-term challenge to dietary CHO availability. That we have observed these diabetogenic alterations in a physically fit population, which is purported to be insulin sensitive yet exhibits high IMTG concentrations (the ‘athlete paradox’) (Goodpaster et al. 2001), supports our contention that they represent an adaptive rather than pathological response. This substantiates our previous assertion that alterations in glucose tolerance and insulin sensitivity associated with dynamic changes to the plasma and/or lean tissue lipid profile are part of a normal co-ordinated adaptation to short-term changes in food availability (Stannard & Johnson, 2004) and perhaps, more specifically, to fluctuations of dietary CHO availability. (...) This short-term alteration is teleologically sound because it limits competition between skeletal muscle and glucose obligate tissues for circulating glucose substrate when its availability becomes limited. Irrespective of a causal relationship, the coupling between IMTG accumulation and reduced insulin sensitivity may also represent a co-ordinated adaptive (non-pathological) response to CHO stress  (Johnson et al. 2003). A concomitant resistance in muscle to the effects of insulin on glucose uptake during CHO stress maintains normoglycaemia and thus the preservation of plasma glucose for use by the CNS and glucose-obligate tissues (Reaven, 1998). Dissociation of insulin action by way of muscle insulin resistance rather than attenuation of insulin secretion means that residual circulating insulin levels can be maintained (Klein et al. 1993), thereby preventing rampant proteolysis (Fryburg et al. 1990), lipolysis (Kather et al. 1985) and perhaps hepatic glucose release, whilst unnecessary uptake of blood glucose by muscle is prevented."

So, from the studies above, we can conclude that:

• FFA supress glucose uptake and oxidation, resulting in muscular insulin resistance, without reducing insulin secretion.
• These effects seem to be dependent on dietary carbohydrate restriction.
• FFA-induced muscular insulin resistance is a physiological response to low availability of glucose. 
• Under normal conditions, this serves to maintain adequate BG levels. When FFA are in excess, there might be a rise in BG levels, because oxidation and release of FFA are not coupled. This leads to insulin resistance in other tissues like the liver (15), consequently failing to control hepatic glucose output.

Blood glucose levels and high carbohydrate diets

Without any biochemical explanation, logic dictates that if we eat a high carbohydrate diet, glucose oxidation pathways are stimulated. This is the opposite of what we observe with carbohydrate restriction, that is, stimulation of insulin signaling. Glucose and insulin both regulate GLUT4 and GLUT1 in muscle cells (16) to increase glucose uptake. The Randle cycle dictates that glucose stimulates its own oxidation and reduces fatty acid utilization. As shown above, glucose reduces PDK and increases PDH. Insulin inhibits lipolysis, further facilitating glucose oxidation. In healthy subjects, a high carbohydrate-low fat diet can improve insulin sensitivity (17, 18). It makes sense, carbohydrates supress fat oxidation and increse glucose oxidation. Overall, there should not be a rise in BG levels on a 24h basis, if anything, we can expect a reduction, because we are utilizing glucose as our main substrate. 

The common ground: calorie restriction

Until now, we have seen that carbohydrates stimulate glucose utilization (insulin sensitivity) and that FFA supress it. How can then a ketogenic diet produce such good results in people with diabetes? Diabetes and MetSyn are characterized by lipotoxicity and glucotoxicity (19). This means that there is an abnormal level of plasma FFA and glucose, produced by PaIR. Insulin cant supress hepatic glucose output, muscle cells do not respond to insulin, and adipocytes liberate FFA in an uncontrolled fashion. In very simple terms, there is an excess of both energy substrates, each one inhibiting the utilization of the other. This scenario can be improved both by restricting fat (thereby increasing glucose utilization) or restricting carbohydrates (increasing fat utilization). In either case, calories must be restricted (directly or indirectly). So, people who show signs of glucose intolerance and switch to a ketogenic diet can improve their BG and insulin levels (by reducing glucotoxicity), but if energy is in excess, BG can start to rise. On the contrary, reducing dietary fat alleviates lipotoxicity, increasing insulin sensitivity. This is why any diet which is calorie restricted, independent of macronutrient composition, produces weight loss and improves glucoregulation. Calorie restriction, by producing a calorie deficit, alleviates both gluco- and lipotoxicity. 

One of the important aspects for dealing with this subject is the fact that glucose intolerance can have many underlying causes. In this manner, a person with autoimmune diabetes may not tolerate carbohydrates as well as a person with only mild PaIR. The fact that PaIR may progress into beta-cell dysfunction (20, 21) can alterate the response to a high carbohydrate diet, and depending on the severity, extreme measures must be taken to achieve normal BG and insulin levels (such as severe calorie restriction). The distribution of body fat can also have consequences on glucoregulation (22). Last but not least, epigenetic changes produced in utero can affect glucose tolerance since the moment we are born (23). 

What is more important, in my opinion, is to address whether hyperinsulinemia causes or potentiates IR, or if hyperinsulinemia results from IR, by a compensatory mechanism. If the first hypothesis holds true, then a ketogenic diet would have an advantage over a low fat-high carbohydrate diet. Desensitization of target cells triggered by the same hormone (homologous desensitization) is a very common characteristic of hormone signaling. In short, high levels of a given hormone reduce the response of the cell to the hormone effects and a reduction in the level of this hormone resets sensitivity. Excess hormone signaling is harmful, so the cell's attempt to restore normality is mediated by reducing its response. This is exactly what happens with insulin: 

• Chronic hyperinsulinemia (in vivo and in vitro) causes a reduction in the number of receptors per cell and glucose transport (24, 25).
• Pre-incubation of 3T3-L1 adipocytes with high levels of insulin and glucose increase PTEN activity, which is correlated with decreased PtdIns(3,4,5)P3 (26). This metabolite is very important for intracellular signaling transduction of insulin.
• Hyperinsulinemia has shown to induce insulin resistance in humans (27).
• Overall, hyperinsulinemia is proposed to be a result and a driver of insulin resistance (28).

Obesity seems to be characterized by an increased amount of insulin being secreted, compared to lean subjects (29). So, while calorie restriction per se is responsible for improved glucoregulation, there might be a short-term benefit in consuming a high-fat ketogenic diet in T2DM and MetSyn patients. As the bodyfat mass and associated hormones regulate, the differences between hypocaloric diets with different macronutrient profiles might be eliminated. This seems reasonable for diet-induced insulin resistance, but not for autoimmune or severe diet-induced glucose intolerance. There seems to be a threshold in which many people cant fully recover their insulin sensitivity with dietary measures. This is where a more integrative and previously uncharacterized approach kicks in (this is the subject of my future post). 

Glucose and cancer

Glucose restriction for cancer treatment seems reasonable given the evidence on the dependence of most types of cancer on glucose for cell growth and proliferation. Unfortunately, the picture is not that simple (30). Although restricting glucose is a good idea, specially for glucose-dependent tumors, the evidence shows that cancer cells also feed on glutamine. More surprinsingly, some types of cancer can grow on fatty acids (31). Restricting glucose reduces insulin levels, which promotes cancer growth. Nevertheless, ideal levels of blood glucose and insulin for treating cancer can only be achieved via calorie restriction. In fact, many supporters of ketogenic diets for cancer often cite the study of Zuccoli et al (32) on the management of glioblastoma. But very few mention what is stated in the study:

"Due to the hyperuricemia the patient was gradually shifted to a calorie restricted non-ketogenic diet, which also delivered a total of about 600 kcal/day. This diet maintained low blood glucose levels and slightly elevated (++) urine ketone levels due to the low calorie content of the diet."

Despite switching to a non-ketogenic (by definition) diet, the patient still showed progress. In my opinion, besides glucose and protein restriction, calorie restriction (and probably fasting) is the dominant factor for achieving success during cancer treatment. 

Summary and key points

• Both energy substrates (glucose and fatty acids) support their own oxidation and inhibit the metabolism of the other.
• A diet high in fat and low in carbohydrates will reduce glucose metabolism and increase fat metabolism. Conversely, a high carbohydrate-low fat diet increases glucose utilization and decreases fatty acid metabolism.
• Increased glucose utilization implies upregulation of glucose membrane transporters and enzymes involved in glycolysis. Additionally, it reduces the activity of enzymes involved in fat metabolism. 
• Increased fatty acid metabolism inhibits key glycolytic enzymes, as well as GLUT membrane translocation. It also interrupts glucose/insulin signaling and stimulates lipolytic enzymes. 
• Chronic hyperinsulinemia, caused by peripheral insulin resistance and energy excess, aggraviates glucose intolerance. Both high glucose and high FFA levels promote this state, by different mechanisms. 
• Under energy balance, a high fat ketogenic diet might produce muscular insulin resistance, reducing glucose tolerance. This should not be compensated by an increase in blood glucose levels. However, if energy intake exceeds calorie expenditure and/or the body utilizes predominantely FFA for energy for extendend periods of time, there can be a rise in blood glucose to non-pathological levels. This is specially relevant if there is little exercise being done (exercise promotes muscular insulin sensitivity) and/or there is an abnormal condition.
• The etiology of glucose intolerance is very important for the proper treatment. Although calorie restriction is the primary solution for obesity/diet-induced insulin resistance, people with autoimmune (both congenital/perinatal or diet-induced)  and beta cell dysfunction should adopt a very low carb approach. 

In the end, the level of carbohydrates proposed by the Jaminet's is in the safe side. The alarmism promoted by some people is not supported. While severely restricting carbohydrates is, in my opinion, the best approach for MetSyn and obesity, once fat mass has reduced, one can tolerate more carbohydrate without problems. If the choice is restricting carbohydrates for life, you should expect a very abnormal response to any carbohydrate (being "safe" or "unsafe"). Nevertheless, lets not forget that the Perfect Health Diet is not a high carbohydrate diet, but a high-fat, low carbohydrate diet. Despite my obvious differences with Paul (33), his dietary advise is very reasonable and his diet is the first I recommend. This template, plus calorie restriction and/or fasting, is the best dietary measure one can implement. Everyone should adjust their individual carbohydrate needs, but in the end, the key is controlling and preventing inflammation. And carbohydrates per se are not inflammatory. 


*Certainly, there are people who are in the extremes of the Gaussian distribution. For these persons, extra measures should be taken. I will write about my approach in the following post. 

No time except for...

...finishing my project. I have to present a draft of my thesis research on January 16th, so I dont have much time to read any paper not related to it.

After that, hopefully I will be able to finish a few pending posts. 

Happy holidays!

Getting fat: the type matters

Traditionally, increased fat mass has been viewed as unhealthy irrespective of the distribution of body fat. With increasing research on the subject, most researchers agree that increased visceral adipose tissue (VAT) is the main determinant of the metabolic disorders associated with obesity, compared to increased subcutaneous adipose tissue (SAT). 

In the last months, there has been a not-so-scientific debate on whether being fat eating a "healthy" diet is better than being thin eating a Western diet. One can be healthy even with an increased body fat mass? Is gaining fat with a given diet different than with another diet?

A study done by Tran et al. (1) caught my attention and motivated me to dig a little further in this topic. The authors found that transplantation of SAT and VAT to the subcutaneous or visceral regions of recipient mice produced remarkable differences on glucose homeostasis, weight and body fat gain. The scheme utilized for the transplants is shown below:

Copyright © 2008 Elsevier Inc. All rights reserved.

The most important effect was noticed in SC-VIS mice, which were transplanted with SAT into the visceral cavity. Compared to other mice, the rate of body weight gain (fat was transplanted "on top" of endogenous adipose tissue) was significantly lower, gaining on average only 63% and 59% (they used two cohorts) of the amount gained by sham-mice at the end of the study. This was irrespective of calorie intake, energy expenditure or heat production. Basal plasma glucose levels were reduced by 15% and plasma insulin levels were reduced by 33% in SC-VIS mice. Compared to sham, SC-VIS mice had 70% lower plasma leptin levels, but adiponectin was also decreased. Intraperitoneal glucose tolerance tests showed that SC-VIS mice had the lowest glucose levels, and insulin sensitivity (assessed by hyperinsulinemic-euglycemic clamp) was higher in this group. Glucose uptake in endogenous SAT was increased in both groups of mice transplanted with SAT, reflecting an increase in insulin sensitivity. Finally, gene expression of adiponectin, resistin and leptin levels were decreased in SC-VIS mice, compared to sham. 

Overall, the study findings were:

• Transplantation of SAT into the visceral cavity produced the most significant results in terms of weight gain, glucose tolerance and adipocytokine levels.
• SC-VIS mice had decreased body weight, decreased body fat percentage, increased percent of lean mass, without significant changes in total energy expenditure or heat production.
• Transplantation of SAT into recipient mice improved insulin sensitivity in the liver and in endogenous SAT. 
• Transplantation of SAT into the visceral cavity decreased average adipocyte area by 38% compared to endogenous SAT, and did not increase the adipocyte's size to that of the endogenous VAT. 
• Adding SAT to the visceral cavity reduced mRNA levels of resistin, leptin and adiponectin, compared to endogenous SAT.

These results suggest a "protective" role of SAT in obesity. There is evidence that insulin resistance correlates with VAT, regardless of bodyweight (2). VAT appears to produce more IL-6 than SAT (3), which correlates with increased macrophage infiltration in VAT compared to SAT (4). Liposuction, despite reducing bodyfat levels, does not improve metabolic markers in the short and long term (5), does not improve insulin sensitivity of muscle, liver or adipose tissue; and doesn't affect levels of C-reactive protein, IL-6, TNFa and adiponectin in diabetic or normal subjects (6). Accordingly, SAT seems to modulate TNFa expression in VAT (7). 

Thus, it seems that it is not weight lost per se which is important for preventing metabolic damage, but the type of body fat lost. Liposuction achieves equal or greater weight loss than lifestyle modifications, but fails to improve metabolic parameters. 

Effect of different diets on body fat distribution

From the above discussion, it is reasonable to think that the best diet is the one that a. decreases body fat mass and b. reduces and/or redistributes fat mass towards SAT.

Active rats fed a ketogenic diet show increased SAT compared to matched carbohydrate-fed rats (8), despite similar body fat levels, although there are contradictory results (9). Weight loss produced either by a high fat-low carbohydrate diet or a low fat-high carbohydrate diet show the same effects on both SAT and VAT (10), suggesting that the macronutrient ratio is not important. This contrasts with a small study which showed that the visceral to subcutaneous fat ratio (V/S) decreased only in the low carbohydrate group (11), compared with the group eating a high carbohydrate diet, even when both diets were hypocaloric. Other authors suggest that ketogenic diets decrease VAT more significantly than high carbohydrate diets (12), although the method used to estimate VAT levels (DEXA) has some predictive problems (13). 

Chaston and Dixon (14) have proposed that acute caloric restriction produces a preferential loss of VAT in the short term and that this effect is seen with modest weight loss. Alternate day fasting (ADF) has also shown to improve body fat distribution in mice, increasing the proportion of SAT vs. VAT and levels of adiponectin, and reducing the levels of leptin and resistin (15, 16). These effects seem to be independent of the diet and body fat loss.

Summing up

Independent of total body fat, there seems to be a protective effect of SAT vs. VAT. This might be the reason why not all obese people develop metabolic syndrome or insulin resistance (17), as healthy obese subjects seem to have less risk for complications than normal-weight subjects with metabolic syndrome (18). Calorie restriction seems to be the most important factor for preventing an increase in VAT, while ADF might provide an additional benefit without weight loss. From the above, we can try to answer the questions proposed: 

One can be healthy even with an increased body fat mass? 

Yes, given a proper body fat distribution (higher proportion of SAT vs. VAT).

Is gaining fat with a given diet different than with another diet?

Possibly. Overfeeding studies done do not control for macronutrient composition, so there is no evidence of a different effect of different diets. However, from the studies available, gaining body fat while implementing ADF might be different than with a Western diet. Moreover, specific nutrients might have different effects regardless of body fat gain, such as fructose (19), although recent evidence do not show adverse effects of overfeeding fructose on visceral fat (20). In either case, having a normal-weight is not protective for cardiometabolic abnormalities (21). This suggests that dietary habits are important even in the absence of weight gain (normal-weight obesity), and that gaining weight with a healthy diet might not be as detrimental as gaining weight with a Western diet.

Tran TT, Yamamoto Y, Gesta S, & Kahn CR (2008). Beneficial effects of subcutaneous fat transplantation on metabolism. Cell metabolism, 7 (5), 410-20 PMID: 18460332

The gut microbiota regulates the metabolic response to fasting

Metabolic adaptation to fasting is an essential mechanism developed by mammals in order to survive. The transition from the fed to the fasted state is tightly regulated. This metabolic shift includes reducing glucose oxidation and storage, and increasing the supply of free fatty acids (FFA) and ketone bodies (KB) to peripheral tissues. Glucose is spared for obligate glucose-consuming cells (such as some neurons, erythrocytes, kidney cells) by FFA's effects on membrane glucose transporters in peripheral tissues, upregulation of lipolytic enzymes and downregulation of glycolytic enzymes. Overall, if extended, fasting will produce a state of peripheral insulin resistance, which implies that skeletal muscle will become desensitized to insulin's effect, thereby reducing glucose transport into cells. However, this scenario differs from that observed during pathological insulin resistance, in which skeletal muscle, liver and adipose tissue are insulin resistant, so there is no control of glucose and FFA levels, leading to glucotoxicity and lipotoxicity.

Healthy humans should be able to fast without problems (metabolic flexibility). If everything is working as supposed to, there should be no problem when switching to a predominantely lipolytic/ketogenic metabolism. How hard the transition to a fasting state is may be a marker of the functioning of intermediate metabolism. 

Crawford et al. (1) tested a basic hypothesis, based on previous findings:

• Germ-free (GF) mice are leaner than conventionally raised (CONV-R) mice, even when GF mice consume more food (2).
• Transplantation of the gut microbiota from obese mice to GF recipients causes a greater increase in adiposity than does a microbiota from lean mice (3).

Overall, there seems to be an alteration in the composition and the microbiome of gut bacteria from obese mice, which increases energy harvest from available food. This is an aberration from its normal function, which is to provide an adequate amount of energy from otherwise indigestable nutrients. If true, then we might expect that this mechanism is beneficial to the host in periods of nutrient deprivation. If not, there would be no obvious evolutionary explanation for this symbiotic relationship. Accordingly,  after withdrawal of nutrients, GF mice die more rapidly that CONV-R mice, even when the rate of body weight loss is the same (4). Because adaptation to fasting involves a shift towards ketogenesis, the gut microbiota might regulate this process.

The gut microbiota regulates ketone body metabolism during fasting

Compared to CONV-D*, GF mice showed 37% lower levels of serum betahydroxybutyrate (BHB) in the fasted state, with no differences in the fed state. Moreover, levels of insulin, glucose, FFA and triglycerides where unchanged. CONV-D mice had increased hepatic triglyceride stores compared to GF mice, difference which was enhanced dramatically with fasting. As expression of Pnpla2 was increased similarly in both CONV-D and GF mice, fasting-induced fatty acid mobilization was not impaired by the absence of gut microbiota. PPARa expression was higher in CONV-D than in GF mice, and the fasting-induced ketogenesis was impaired in CONV-D PPARa-/- mice. 

The liver produces BHB for utilization in peripheral tissues. It lacks the key enzyme 3-oxoacid CoA transferase, so is incapable of oxidizing ketone bodies. When fasted, GF mice had 50% less concentration of liver BHB compared to CONV-D mice. Expression of Fgf21 and Hmgcs2, both targets of PPARa which stimulate ketogenesis, was lower in fasted GF mice compared to CONV-D mice. 

These results suggest that the gut microbiota stimulates ketogenesis during fasting by a PPARa-dependent mechanism. Additionally, it promotes hepatic triacylglycerol synthesis and storage. 

There are two possible ways of generating acetyl CoA in the liver during a fast: from acetate produced in the gut and from oxidation of fatty acids from adipose tissue. In GF mice, the only source is the latter. Cecal acetate levels were very low in fed GF mice and 20-fold greater in CONV-D mice. During fasting, these levels were reduced in CONV-D, but remained significantly higher compared to GF mice, which showed no reduction. 

One unexpected result was the change in the microbiota composition induced by fasting. It was found that there was a significant increase in the proportion of Bacteroidetes and a significant reduction in the proportion of Firmicutes. Previous studies have found a correlation between a reduction in Bacteroidetes and an increase in Firmicutes with obesity (the high Firmicutes/low Bacteroidetes hypothesis) (5, 6). 

The gut microbiota influences myocardial metabolism

Analysis of the myocardial transcriptome of CONV-D and GF mice revealed an enrichment in the ketone body metabolic pathway in both groups, compared to their PPARa -/- counterparts. Expression of genes involved in fatty acid and ketone body metabolism were increased with fasting in both groups, but Oxct1 gene expression was higher in CONV-D mice. Conversely, an increased Glut1 expression was only observed in GF mice. 

The rate of glucose oxidation was significantly increased in isolated working hearts of GF mice, without alteration of fatty acid oxidation. In the absence of BHB, glucose utilization was also significantly greater. Glycogen levels were reduced in the myocardium of fasted GF mice compared to CONV-D mice, and there were no significant differences in heart rate, cardiac hydraulic work, mitochondrial morphology or number, or mitochondrial respiration. 

The shift towards a ketogenic metabolism in the myocardium is one hallmark of adaptation to fasting, as BHB is more energy efficient than glucose. So in periods of deprivation of nutrients, the myocardium maintains its normal functioning by using BHB instead of relying in glucose. This adaptation is impaired in GF mice.

The gut microbiota affects myocardial mass

Heart weight was reduced in fasted GF mice compared to CONV-D mice. Training elicits an hypertrophic response in the heart. However, this response was blunted in the absence of gut microbiota, as the hearts of trained GF mice remained smaller than the hearts of trained CONV-D mice. This correlated with alterations in a subset of pathways, which included ketone body metabolism. Administration of a ketogenic diet rescued heart weight in GF mice and shifted the myocardial transcriptome toward ketone body metabolism.

These results suggest that the gut microbiota is an important component for cardiovascular health, and that ketone bodies represent an essential substrate for the heart. 

Summing up

This is one of the most interesting studies that I have read lately. It provides a new template for the relationship between metabolism and gut microbiota, and shows the importance of gut bacteria for the normal response to fasting. I have summarized the findings of the study in the following figure (my interpretation):

The gut microbiota regulates ketogenesis during fasting. Fasting induces an increase in the proportion of Bacteroidetes and a reduction in the proportion of Firmicutes. These changes promote the production of acetate, which serves as substrate for hepatic acetyl CoA synthesis. The gut microbiota also stimulates hepatic triglyceride stores, providing another source of energy during fasting. The increase in acetyl CoA levels stimulates ketogenesis by a PPARa-dependent mechanism, increasing serum BHB levels. The elevated concentration of BHB levels supplied to the heart promotes the shift towards a ketone body-based metabolism, and inhibits glucose oxidation. Myocardial ketone body metabolism maintains myocardial mass and the normal hypertrophic response to exercise.

* CONV-D (conventionalized) mice were transplanted with distal gut microbiota from CARB-fed CONV-R lean mice. 

Crawford PA, Crowley JR, Sambandam N, Muegge BD, Costello EK, Hamady M, Knight R, & Gordon JI (2009). Regulation of myocardial ketone body metabolism by the gut microbiota during nutrient deprivation. Proceedings of the National Academy of Sciences of the United States of America, 106 (27), 11276-81 PMID: 19549860

Fecal bacteriotherapy

Proper gut microbiota establishment begins in the moment we are born and is shaped by lifestyle and environmental factors in subsequent years. In some cases, the degree of dysbiosis is so severe that there is not turning back and practical dietary/lifestyle recommendations are useless.

Fecal bacteriotherapy

The logic behind this intervention is simple: it tries to "reset" the gut microbiota. It has shown promising results in intestinal bowel disease (IBD) and resistant Clostridium difficile infections. The following protocol is taken from Silverman et al (1). My intent is to facilitate information, not to encourage the realization of this protocol without medical supervision. Interested persons should consult with their doctors before doing any procedure of this nature. Donors and recipients should be examined carefully before the intervention. The complete set of tests can be consulted in the mentioned study.

Pretreatment

Recipients are initiated on maintenance therapy with oral Saccharomyces boulardii (probiotic), 500mg orally, twice per day. Metronidazole (500mg/3 times per day, PO) or vancomycin (125mg/4 times per day, PO) are also used. Both are antibiotics normally used against C.difficile infections.

Equipment

- 1 bottle of normal saline (200mL)

- 2 standard 2 quart enema bag kits (available at drug stores)

- 3 standard kitchend blenders (1L capacity) with markings for volume

Procedure

- Vancomycin/metronidazole should be stopped 24-48 hours before procedure.

- S.boulardii should be continued during the transplant and 60 days afterwards.

1.     Add 50mL of stool (volume occupied by solid stool) from the healthy donor immediately prior to administration (< 30 minutes) to 200mL of normal saline in the blender.

2.     Mix until getting a "milkshake" consistency.

3.     Pour mixture (approximately 250mL) into the enema bag.

4.     Administer enema to the recipient following the kit instructions. The patient should hold the infusate as long as possible and lie still as long as possible on his/her left side to prevent the urge of defecation. The procedure should be ideally performed after the first bowel movement.

5.     If diarrhea recurrs within 1 hour, the procedure may be immediately repeated.

Modifications and perspectives

This procedure was made to treat C.difficile infections. Accordingly, the antibiotics and the probiotic used aimed to eliminate C.difficile from the gut. However, there are certain modifications which can be useful for treating severe dysbiosis. First, broad-spectrum antibiotics can be used to wash out most bacterial species and reduce colonization resistance. In addition, utilization of probiotics such as Bifidobacteria or Lactobacilli during and after the treatment should help preventing colonization by enteropathogenic species. Why Bifidobacteria? The use of broad-spectrum antibiotics increases the risk for colonization of enteropathogens. Bifidobacteria competes and prevents colonization by these pathogens directly and indirectly, via production of antibacterial molecules (2). In addition, dysbiosis is characterized by low levels and expression of Foxp3+ Tregs, which compromises immune tolerance and promotes inflammation. Oral administration of B.infantis has been shown to increase expression of Foxp3+ and IL-10 in peripheral blood and to drive maturation of dendritic cells towards a regulatory phenotype (3), and certain strains of Bifidobacteria are capable of modulating the plasticity of Th17/Treg populations in human PBMCs (4). On the other hand, Lactobacilli has also shown protective properties (specially against vaginal infections) (5) and competes with enteropathogens for adhesion on intestinal epithelial cells (6). Importantly, the effects over Treg induction and T cell differentiation differ between strains from the same species. I should address this issue in future posts. One thing that is not emphasized in the above protocol is the importance of diet for maintaining a correct microbiota. This, in my opinion, is key to success.

It is worth noting that because of the nature of the procedure, the microbiota of recipient subjects is altered and reduced, but not completely eliminated such as seen with studies in fecal transplantation. The utilization of fecal transplantation in humans is promising and should result in better outcomes. Indeed, positive preliminary results from the FATLOSE trial (7, 8) have been recently published in which patients with metabolic syndrome improved insulin resistance and lipid profiles after feces infusion from healthy donors. The positive results seem to be correlated with increases in colonic butyrate concentrations. These results fit nicely with the ones found previously with fecal transplantation in obese mice.

Turnbaugh et al (9) found astonishing differences in the microbiome of obese mice, compared to lean mice (greater abundance of Firmicutes). Metagenomic analysis revealed that the obese microbiome is enriched for EGT (environmental gene tags) encoding many enzymes invoved in the break down of otherwise indigestable dietary polysaccharides. These included KEGG pathways for starch/sucrose metabolism, galactose metabolism and butanoate metabolism. Increased concentrations of butyrate and acetate were also observed, as the fact that obese mice were able to harvest more energy compared to lean mice (assessed by less energy remaining in feces by bomb calorimetry,). Despite equal amount of food consumed in both groups, colonization of lean mice with obese microbiota led to an increase in bodyfat percentage of approximately 47% after two weeks. The potential for fecal bacteriotherapy in the treatment of several diseases has been observed in different animal models of inflammatory and autoimmune diseases.

Thus, it seems possible that future therapies for obesity, metabolic syndrome and other inflammatory/autoimmune conditions will aim to modulation of the gut microbiota.

Silverman MS, Davis I, & Pillai DR (2010). Success of self-administered home fecal transplantation for chronic Clostridium difficile infection. Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association, 8 (5), 471-3 PMID: 20117243

Glucose restriction and TSC

Recently, zooko asked me about my opinion on a recent study just published (1).

Background

TSC1 and TSC2 are a pair of tumor supressor genes, which relevance lies in the inhibition of mTORC1 activity. mTOR (the mammalian target of rapamycin) is a master regulator of cell proliferation, cell growth, cell motility, cell survival, protein synthesis and transcription. Because of this, dysregulation of the mTOR pathway is seen in many cancers (2).

mTOR forms two complexes, mTORC1 and mTORC2 ("rapamycin-insensitive"), which respond to different stimuli. TSC2 has a GAP (GTPase activating protein) domain that stimulates the GTPase activity of Rheb. GDP-Rheb is inactive, while GTP-Rheb is active. By this mechanism, TSC2 accelerates the hydrolysis of GTP, inactivating Rheb. Active Rheb is a potent activator of mTORC1. The interplay between these proteins is shown below (3):

In response to growth factors, Akt phosphorylates TSC2 directly on four or five residues (Ser939, Ser981, Ser1130, Ser1132 and Thr1462). Phosphorylation of TSC2 by Akt impairs its ability to inhibit Rheb, thereby blocking the inhibitory effect of Rheb on mTORC1. Other mechanisms proposed to explain contradictory experimental results include the action of PRAS40 and binding of 14-3-3 to phosphorylated TSC2.

The most conserved pathway for Akt activation is the PI3K/Akt pathway. Insulin/IGF-1 binding to the insulin receptor produces phosphorylation of its cytosolic domain, promoting the binding of IRS (insulin receptor substrate) by its PTB (phosphotyrosine binding) domain. This promotes the association (by SH2 domains in the p85 regulatory subunit) and activation of PI3K. PI3K phosphorylates phosphatidylinositol-4,5-biphosphate (PtdIns(4,5)P2), producing PtdIns (3,4,5)P3. PtdIns (3,4,5)P3 binds to the PH domain of Akt and promotes its translocation to the plasma membrane. PI3K-dependent kinase 1 (PDK1) then phosphorylates Akt on Thr308 and PDK2 phosphorylates Ser473. Both phosphorylations activate Akt. Phosphorylated Akt, as previously mentioned, phosphorylates and inactivates TSC2 and PRAS40 promoting mTORC1 activation. The activation of the PI3K/Akt pathway has many downstream cellular events promoting cell survival and proliferation, which include inactivation of several proapoptotic factors (BAD, procaspase-9 and Forkhead transcription factors), activation of antiapoptotic factors (CREB), activation of IKK, inactivation of p53, among other. The net result is promoting proliferation and cell survival, hallmarks of cellular malignancy development and progression.

Discussion of the study

The basis for the utilization of glucose restriction for treating TSC related tumors can be easily inferred from the above explanation. By restricting glucose, insulin signaling is reduced, so is activation of mTORC1. mTORC1 also upregulates HIF1a, promoting aerobic glycolysis and lactate production (4). Thus, glucose restriction should promote apoptosis, specially in TSC1/TSC2-null cells.

What the authors basically did was treating Tsc2-/- mice with a carbohydrate-free diet (CF) and a Western Diet, alone or combined with 2-deoxyglucose (2-DG). Preliminary in vitro studies showed that Tsc2-/- cells were sensitive to glucose restriction and 2-DG in an additive manner. Contrary to what was expected, in vivo experiments showed that tumor size and growth rate were highest in the CF group and 2-DG supressed tumor growth independently of diet. These results also contradicted the observed standard uptake values (SUV) during the FDG-PET scan (presented as the maximum SUV within each tumor). Theoretically, as these tumors are sensitive to glucose deprivation, there should be a correlation between glucose uptake (measured by uptake values of FDG) and tumor size (increased tumor size should show increased SUV). However, there was no correlation between these parameters, as the CF+2-DG group showed the minimum mean SUV but the largest tumor size. What can be fueling tumor growth? Ketone bodies? An in vitro assay showed that addition of either acetate or beta-hydroxybutyrate to Tsc2-/- cells increased cell confluence and reduced the number of non-viable cells (assessed by trypan blue), compared to glucose alone. To further complicate things, ketonemia was not developed in CF mice, but beta-hydroxybutyrate levels were higher with the Western +2-DG diet. Testing the effects of fatty acids in vitro showed that palmitic acid induced necrosis and oleic acid induced proliferation. This correlated with the histologic analysis of CF mice. Addition of rapamycin reduced cell-size, in contrast with 2-DG, which decreased proliferation. Finally, there was increased activation of mTORC1 (measured by phospho-S6) and low levels of phosphorylated Akt (secondary to feedback inhibition) in all groups, with no differences between groups.

Interpretation

First, the results confirm the potent anti-tumor activity of 2-DG. Second, the CF group failed to establish ketosis, and the Western group had increased levels of beta-hydroxybutyrate, as well as reduced tumor size. This (despite the observed growth-promoting properties of acetate and beta-hydroxybutyrate in vitro, see below) can be interpreted as an inhibitory effect of ketonemia on cancer growth. The comparison of glucose and beta-hydroxybutyrate levels is shown below:

The diet which resulted in lower glucose levels and higher ketone bodies was associated with reduced tumor size, and the diet which produced greater glucose levels and lower ketone bodies was associated with increased tumor size. The results observed in vitro with Tsc2-/- cells and ketone bodies suggest that in this cell line, it is necessary an additional anti-glycolytic factor to control tumor growth (2-DG), because these (and other) cancer cells seem to be capable of metabolizing ketone bodies (5, 6). This underscores the importance of the phenotype of the tumor being treated, an important factor that is not taken into account by some "low-carb" advocates who think that restricting dietary glucose will magically cure all cancers.

Another important factor to take into consideration is that mice were not calorie restricted, and more importantly, that the CF diet was high in protein. Glutamine is a major substrate that can fuel cancer cells (7, 8). On a more general level, aminoacids are potent stimulators of mTORC1 (9, 10). This is specially relevant for this model, because excess aminoacids can by-pass the inhibition of the PI3K/Akt pathway by promoting Rheb co-localization with mTORC1 (11), activating mTORC1 in the abscence of TSC2 (12). Ketone bodies increased ATP levels in Tsc2-/-in vitro. This reduces AMPK activity. AMPK inhibits mTORC1 activity by TSC2 dependent and independent mechanisms (possibly by phosphorylation of Raptor) (12). 2-DG also increases intracellular AMP levels (activating AMPK), which would explain the benefits of its utilization observed in this model (13). Supporting the role of AMPK as a target for cancer treatment, the combination of metformin and 2-DG seems to be more toxic to cancer cells than either by itself (14). Interestingly, AMPK activity was not changed in response to 2-DG in this model, which suggests that there are other mechanisms mediating the anti-proliferative effect of 2-DG.

Summing up

Cancer is a very complex disease which treatment has to be personalized depending on the phenotype. With the increase knowledge in cancer molecular biology and genetics, therapies should be designed depending on specific markers evaluated. This complexity explains why not all cancers can be treated just by restricting glucose and making such statement is ludicrous. Besides calorie, glucose and protein restriction, compounds such as 2-DG and metformin show promising effects for controlling most types of cancer.

Jiang X, Kenerson HL, & Yeung RS (2011). Glucose deprivation in Tuberous Sclerosis Complex-related tumors. Cell & bioscience, 1 (1) PMID: 22018000