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

Is phytate really a problem?

As mentioned in a previous post, there is increasing evidence of adaptation to gluten consumption by humans. This adaptation is not genetic, but symbiotic. It appears that we have developed new symbiotic relationships with specific microorganisms to help us degrade gluten, and by doing so, being able to exploit an unnatural food source. 

Aside from resistance to degradation by mammalian enzymes and creation of neo-epitopes from partial gliadin digested peptides, one common reason given for avoiding gluten by paleo advocates is its phytate content (1, 2). Phytate is an anti-nutrient which binds to and form complexes with proteins, lipids, carbohydrates, and metal ions (zinc, iron, calcium and magnesium) thereby reducing their bioavailability. Phytate is the common name for myo-inositol-(1,2,3,4,5,6)-hexakiphosphate (InsP6). 

scienceblogs.com

From its chemical structure, we can see that it is basically a myo-inositol with six phosphate groups. The ability to degrade InsP6 is conferred by phytases. There are three types of phytases, namely, 3-phytase, 5-phytase and 6-phytase. The differences between these phosphatases is the position on the inositol ring at which the initial attack of a phosphoester bond takes place. Thus, attack by different phytases produce different isomers. Phytase production and activity in humans is relatively low (mainly in the small intestine) (3), so the greatest source of phytases is the gut microbial community . 

Gut flora phytase activity

Of the Bifidobacteria species which predominate in the human gut, the B. catenulatum group (B. catenulatum and B.pseudocatenulatum) is the most common. Haros et al (4) examined the InsP6 degrading capacity of B.pseudocatenulatum ATCC2919, isolated from the human gut. It was found that B.pseudocatenulatum is able to degrade InsP6 in sequential dephosphorylations (starting in the 6-position of the myo-inositol ring, followed by the 5-position). The solubility of mineral chelates of myo-inositol phosphates is related to the number of phosphates per molecule. InsP6 and InsP5 have adverse effects on mineral absorption. On the contrary, breakdown products with 1,2,3-grouping interact specifically with iron, increasing its solubility and preventing its ability to catalyse hydroxyl radical formation. Overall, the mineral-binding strength to inositol phosphates  becomes progressively lower when phosphate are removed from the molecule (with the exception of the 1,2,3-grouping mentioned above). B.pseudocatenulatum also showed selective adhesion to Caco-2 epithelial cells and tolerance to increased concentrations of bile, which reflects its adaptation to the human gut. A previous study (5) found that B.infantis is able to degrade 100% of InsP6, producing InsP3 as the main product. The optimal pH for the phytase activity of B.infantis was 6.0-6.5, with an activity of 51.2% at 37C; similar to that observed for B.pseudocatenulatum. Other Bifidobacteria species present in the human gut have also phytase activity, although to a lesser extent.

InsP6 antinutrient effect

Typically, InsP6 and fiber occur together in whole foods. This is problematic for analyzing the antinutrient effect of InsP6 as there is evidence that fiber also reduces mineral bioavailability (6). When given alone in animal models, InsP6 does not show toxic effects on bone minerals (7):

This suggests that its the combination of fiber and InsP6 which causes the antinutrient effect observed. 

The type of fiber seems to be important on mineral bioavailability. The addition of FOS to a diet high in InsP6 improves cecal absorption of minerals and stimulates bacterial hydrolysis of InsP6 (8, 9), counteracting the negative effects of high doses of InsP6. Inulin has also shown to improve calcium balance and absorption (10). The importance of the fiber type on the effects of phytic acid is highlighted by a study in which healthy women following the recommended daily intake of fiber-rich wheat bread (300g/day) showed impaired iron status independent of the phytic acid content (11).

Anti-cancer properties

InsP6 is a broad-spectrum antioneoplastic agent in vitro and in vivo (12). Structurally, InsP6 is similar to D-3-deoxy-3-fluoro-ptdIns, a potent PI3K inhibitor. Accordingly, InsP6 is able to inhibit PI3K and ERK phosphorylation (13), thereby inhibiting AP-1 activation. InsP6 has also been shown to activate PKC delta and decrease phosphorylation of Erk1/Erk2 and Akt, causing upregulation of p27-Kip1 and reduction of pRb phosphorylation (14). Other protective effects include the induction of apoptosis by inhibiting the Akt-NFkB pathway and increasing cytochrome C release (15), downregulation of constitutive and ligand-induced mitogenic and cell survival signaling (showing different effects on ERK1/2, JNK1/2 and p38 in response to different mitogens) (16), its antioxidant effect (17), enhancement of NK cell activity (18), modulation of expression of TNF-alpha and its receptors genes (19), inhibition of angiogenesis (20) and metastasis, by modulation of integrin dimerization, cell surface expression and integrin-associated signaling pathway (lack of clustering of paxilin and reduced FAK autophosphorylation) (21, 22). Utilization of InsP6 has been shown to offer some benefits during chemotherapy (23) and future trials are on their way.

Are whole grains inherently unhealthy?

Because whole-grains and legumes are high in phytic acid, it is plausible to hypothesize that intake of these foods will reduce to some extent the risk of developing cancer. Whole-grain intake has been associated with reduced risk of cancers (24, 25) as well as intake of legumes (26). However, some studies have found no association (27, 28). Because of the nature of these studies, it is not possible to draw causative conclusions. Most people eating the supposedly healthy foods have low intakes of harmful foods, so the decreased risk in some studies might be due to the exclusion and not the inclusion of some foods. In either case, most studies have not observed an increased cancer risk associated with these foods*. Other food sources rich in phytic acid include nuts and cocoa. 

Conclusions

The dangers of phytic acid have been overestimated. Contrary to popular the paleo belief, phytic acid might be beneficial in small doses and might have anticancer effects. As seen with gluten degradation by Rothia species, the phytase activity present in some exclusive human Bifidobacteria shows that adaptation to wheat/grains is indeed happening. Once again, the microbiota plays a dominant role.

From epidemiological data, foods with high phytate content are not associated with increased risk for several chronic diseases. As association doesnt means causation, we cannot conclude that whole-grains are healthy but we cant also conclude that whole-grains are unhealthy. With the increasing attention to paleolithic and similar diets, it is of utmost importance that all evidence is critically analyzed and reviewed. Making unsupported statements and cherry-picking data would only cause rejection by scientists. Dogma is not good in science (or in anything else, for that matter).

I dont recommend whole-grains and legumes because there are foods more nutritious, as well as because whole-grains and legumes are very high in carbohydrates. The potential benefits of phytate can be obtained by eating other phytate rich foods, such as nuts and cocoa; as well as soluble fiber and oligosaccharides as the main dietary fiber type. The problem with high levels of phytate is only relevant when the diet is deficient in micronutrients and essential food sources. Finally, maintaining a proper gut flora is essential for phytic acid metabolism and adequate mineral absorption. 

*Any evidence of a significant increased risk from these foods would be greatly appreciated.

Haros M, Carlsson NG, Almgren A, Larsson-Alminger M, Sandberg AS, & Andlid T (2009). Phytate degradation by human gut isolated Bifidobacterium pseudocatenulatum ATCC27919 and its probiotic potential. International journal of food microbiology, 135 (1), 7-14 PMID: 19674804

Haros M, Bielecka M, Honke J, & Sanz Y (2007). Myo-inositol hexakisphosphate degradation by Bifidobacterium infantis ATCC 15697. International journal of food microbiology, 117 (1), 76-84 PMID: 17462768

Bifidobacteria, butyrate and carbohydrates

In a previous post, john asked:

Regarding your old post on ketogenic diet and microbiota, why do you think bifidobacterium decreased on low carb? I would generally guess this is a negative...?

I cited two studies on low carbohydrate dieting and gut microbiota composition, one by Duncan et al (1) and the other by Brinkworth et al (2). They showed a negative effect of reducing carbs on gut flora, measured by species composition (16S RNA) and SCFA. They both analyzed fecal samples. In general, fecal samples are reliable and make easier to study colonic SCFA metabolism. However, they are an indirect method of quantification. Of the three main SCFA produced in the colon, only acetate has shown a correlation between fecal concentration and absorption (3):

Copyright © 2011 by the American Society for Nutrition

The data shows a negative correlation (r=-0.834) between acetate absorption from an infusion and fecal acetate concentration. This means that the fecal concentration of acetate might reflect absorption rather than production, in an inverse manner (less acetate in fecal samples equals more absorption). In this study, neither propionate or butyrate showed a correlation between absorption and fecal concentration. 

SCFA in the Duncan et al. study

Acetate, butyrate and propionate concentrations in fecal samples from the Duncan et al. study are shown below:

SCFA concentrations (mM) for fecal samples. M=Maintenace; HPMC= High-protein, moderate-carbohydrate; HPLC = High-protein, low-carbohydrate. Mean values.

As the intake of carbohydrate decreased, there was a parallel reduction in all three SCFA. 

SCFA in the Brinkworth et al. study

Acetate, butyrate and propionate concentrations in fecal samples from the Brinkworth et al. study are shown below:

Fecal SCFA concentrations (mM) after 8 weeks of either a low carbohydrate (LC) or high carbohydrate (HC) diet. Mean values. 

As seen in the Duncan et al. study, after 8 weeks with a low-carbohydrate diet, SCFA concentrations were reduced, although not as drastically. 

Analysis and interpretation of the data

Both studies show a clear correlation between carbohydrate intake and SCFA concentration in fecal samples. The magnitude of the changes between individual SCFA might be due to differences in the intervention time (4 weeks vs. 8 weeks). 

As shown by Vogt and Wolever (see above), acetate concentration in fecal samples reflect more precisely acetate absorption rather than production. Thus, lower fecal acetate levels with reduced carbohydrate reflect more acetate absorption (or utilization, see below). 

Most focus has been given to the apparent reduction in butyrate levels, which may compromise colonic health. In this regard, low carbohydrate diets might be detrimental for colonic health because of reduced butyrate production. For assessing the validity of this statement, we must look at colonic butyrate metabolism. 

Colonic butyrate metabolism 

Approximately, 95% of the butyrate produced in the colon is absorbed. This is why fecal concentrations are not a good guide to production rates: a very high proportion of the SCFA is taken up by the colonic mucosa (4). Butyrate is produced from two molecules of acetyl CoA, yielding acetoacetyl CoA, which is further converted to finally butyryl CoA. This metabolite can be converted to butyrate via butyrate kinase or butyryl CoA:acetate CoA transferase.

Butyrogenic substrates include starch, inulin and xylan. But certain species are capable of producing butyrate from acetate. Synthesis of butyrate from acetate is performed via the butyryl CoA:acetate CoA transferase pathway, which seems to be the most prevalent route of butyrate synthesis by human gut bacteria (5). So, while glucose is needed for butyrate synthesis, acetate seems to be the main substrate for butyrate formation. The predominance of butyrate synthesis from the acetate dependent pathway might reflect a selective advantage for bacteria which transform acetate to butyrate in the colon, where acetate concentrations are high. 

Overall, the reduction in acetate and butyrate fecal concentrations may be translated to increased absorption and reduced excretion. Butyrate can be synthesized from acetate, which reduces the concentration of both SCFA in feces. The determined Km for butyrate transport in the colon has been found to be 14.8 +/-3.6 mM (6) and 17.5 +/- 4.5 mM in the proximal colon (7). The apparent saturation kinetics showed by butyrate transport across the colonic luminal membrane could further explain the results seen in the studies mentioned above: increasing the carbohydrate content in the diet would augment the number of glucose-dependent butyrogenic bacteria, increasing the colonic production and concentration of butyrate. Because transport of butyrate is saturable, excess butyrate is excreted, producing increased levels in feces. 

The case for Bifidobacteria

The change in Bifidobacteria concentrations after the low carbohydrate diet is due to the presence of an important number of bacteria capable of degrading glucose/starch. In this scenario, reduced carbohydrate availability would reduce the number of total Bifidobacteria (at least certain species). This does not mean that this is bad per se. It is important to determine the specific Bifidobacteria which are responsive to diet. For instance, B.longum seem to be capable of catabolizing not only dietary oligosaccharides, but also glycoproteins and glycoconjugates from the host; as well as nucleotides (8). Moreover, gut Bifidobacteria (as shown by the genomic analysis of B.longum), are capable of adapting to different carbohydrate substrates depending on their availability (9). In addition, metabolic-crossfeeding occurs between Bifidobacteria and other species. For example, E.hallii, a butyrogenic bacterium, is unable to grow on pure starch by itself. Co-culture of this bacterium with B.adolescentis stimulates its growth and butyrate synthesis, paralleled by a reduction in lactate levels (10). The scheme is pretty simple: B.adolescentis is capable of fermenting starch, producing lactate which serves as substrate to E.hallii. Other lactate-independent mechanisms of cross-feeding have also been observed in FOS and oligofructose-only co-cultures of B.longum with Roseburia intestinalis or Anaerostipes caccae, which are cabaple of producing butyrate and consuming acetate (11).

Because of its complexity, the specific mechanisms by which certain Bifidobacteria could be beneficial are unknown, although there is evidence of health benefits from increasing gut Bifidobacteria (12, 13, 14). There are some issues with interpreting the evidence in this topic:

•  Many authors don't determine the exact species being studied (take all Bifidobacteria as a group).
• Supplementation is done with different strains and the long-term effects are not known, because bacteria supplemented via diet are treated as allochthonous. 
• Genomic inspection has shown that Bifidobacteria are metabolically very flexible. Adaptation to substrate variations might take longer than 8 weeks. 
• There is metabolic-crossfeeding occuring between bacteria. This is a highly complex network of connections for which we are only starting to get an initial picture. 

Having this in mind, I cant assure either that a low carbohydrate diet is not harmful to the gut microbiota. As far as the evidence goes, we can only speculate and formulate hypotheses. And useful hypotheses should be based on logic and evolutionary inference. We should ask not only "how" but also "why". In this case, we are not going to focus on the "how" but on the "why"; to put it formally, why an increased intake of starch is associated with an increase in Bifidobacteria? What is the evolutionary basis?

One important protective role of Bifidobacteria is preventing colonization of enteropathogens by reducing their adhesion to intestinal epithelial cells. This has been shown directly for E. coli and S. typhimurium (15). Other commonly problematic Enterobacteriaceae include Klebsiella and Shigella. Growth of these pathogens is stimulated by high glucose-low oxygen conditions. The selective advantage of having responsive Bifidobacteria in the gut might be protection. As increased glucose concentrations favor the development of an adequate environment for growth of these pathogens, there has to be a mechanism by which the composition of the normal microbiota is maintained. So there is a parallel increase in Bifidobacteria with increasing concentrations of dietary carbohydrates to restrain colonization of pathogenic anaerobes. The fact that certain species of Bifidobacteria can metabolize different oligosaccharides and adapt to the substrate availability supports this hypothesis. I might elaborate more on this in subsequent posts. 

Conclusions

Low carbohydrate diets seem to reduce the fecal concentration of SCFA in the short term. Some adaptation seems to occur, judging by the differences between the study periods (4 weeks vs. 8 weeks). Fecal concentrations of SCFA are not good indicators of SCFA colonic production. Conversely, they rather reflect excretion (butyrate) and absorption (acetate). Butyrate can be produced from different substrates, of which acetate is the main precursor in the human gut. There is a reduction in the levels of Bifidobacteria detected in stool samples, proportional to the decrease in carbohydrate in the diet. Although no individual species where identified, studies have shown that Bifidobacteria are capable of adapting to substrate availability and cross-feed with other bacteria. The evolutionary basis for increased Bifidobacteria in response to sugar might involve a protective mechanism against colonization of enteropathogenic bacteria, such as E. coli, Klebsiella, Shigella and Salmonella.

Duncan SH, Belenguer A, Holtrop G, Johnstone AM, Flint HJ, & Lobley GE (2007). Reduced dietary intake of carbohydrates by obese subjects results in decreased concentrations of butyrate and butyrate-producing bacteria in feces. Applied and environmental microbiology, 73 (4), 1073-8 PMID: 17189447

Brinkworth GD, Noakes M, Clifton PM, & Bird AR (2009). Comparative effects of very low-carbohydrate, high-fat and high-carbohydrate, low-fat weight-loss diets on bowel habit and faecal short-chain fatty acids and bacterial populations. The British journal of nutrition, 101 (10), 1493-502 PMID: 19224658

Rothia to the rescue

Gluten is problematic. Almost every paleo advocate agrees that wheat should be restricted in the diet because gliadin peptides generated by the uncomplete digestion of gluten produces highly reactive epitopes, which then can trigger a T-cell response in the gut. 

The main issue with the "gluten is bad for everyone" meme is that not all people develop gluten sensitivity or celiac disease. Many can tolerate great doses of wheat-based foods for years without serious health consequences. This is often attributed to lack of an environmental trigger which increases susceptibility to gliadin peptides (ie. inflammation). 

A recent paper (1) has found that two strains from the genus Rothia, namely R.mucilaginosa and R. aeria are capable of metabolizing gluten. These are oral microorganisms.

After isolation on a gluten agar media, these microbes were capable of hydrolizing YPQ tripeptides (which occur with high frequency in gluten sequences) and KPQ. Moreover, R.aeria degraded gliadin in vitro: 

SDS-PAGE of aliquots from the incubation mixture

The arrow shows the major protein constitutent in the gliadin mixture. As it can be seen, gliadin was progressively degrated (lanes 2-7, figure A). Shorter time intervals (lanes 2-7, figure B) show that almost 50% of gliadin was degraded in 30 minutes. Boiling bacterial suspensions abolished degradation (lanes 8 and 9, figure B). This suggests enzyme denaturation. Lanes 10-11 and 12-13 served as negative and positive controls, respectively. 

Proteolytic degradation of two problematic peptides (a-gliadin derived 33-mer and y-gliadin derived 26-mer) was compared between mammalian enzymes (pepsin, trypsin, chymotrypsin) and R.aeria:

RP-HPLC of sample aliquots

As it can be seen, chromatograms from all mammalian enzymes show the same pattern at 0 and 24h, showing no digestion of the peptides (A-C). In contrast, the sample containing R.aeria (WSA-8, figure D) showed the presence of different peaks earlier in the chromatogram, representing degradation fragments which elute earlier. At 2 hours, the peptide was completely degraded. 

These results show that enzymes present in R.aeria are capable of degrading gliadin and two peptides (33-mer and 26-mer) which are resistant to mammalian enzymes. Analysis by Mass Spectrometry determined that cleavage was made after QPQ and LPY for R. aeria and XPQ and LPY for R.mucilaginosa (X denotes any aminoacid). This is important because these tripeptides are part of the immunogenic epitopes contained within the 33-mer (glia-a9, glia-a2) and 26-mer peptides (glia-y2):

glia-a9: LQLQPFPQPQLPY

glia-a2: PQPQLPYPQPQLPY

glia-y2: PFPQQPQQP / PYPQQPQQP

It is worth noting that cleavage from both Rothia strains was observed also after Q residues along the 26-mer sequence. Repeated Q residues (along with P residues) are responsible for resistance to proteolysis by mammalian enzymes. 

Zymography at pH 7.0 showed that the putative gliadin-degrading enzymes had a molecular weight of approximately 75kDa and 70kDa for R.mucilaginosa and R.aeria, respectively. Further analysis on the activity of these enzymes at different pH revealed that enzymes from R.mucilaginosa were completely inactive at pH 3.0, while R.aeria maintained a weak enzymatic activity. The optimal pH determined was 7.0, and substrate hydrolysis rates declined in parallel with decreasing pH values from 7.0 to 4.0. At pH 3.0, R.aeria showed a much slower activity, while at pH 2.0, activity was completely abolished. 

Summary

- R.mucilaginosa and R.aeria were capable of degrading gliadin and immunogenic peptides in vitro.

- The enzymes present have an approximate molecular weight of 70-75kDa. 

- Degrading activity of R.aeria was maintained at pH 3.0. 

- Both species are normal colonizers of the oral cavity and other areas (R.mucilaginosa).

From the discussion (my bolds):

"R. aeria (...) is an oral colonizer [31]. R. mucilaginosa also primarily colonizes the oral cavity [33] but has furthermore been isolated from other body sites, including the upper respiratory tract and the duodenum [34,35,36]"

"Bacterial speciation of 2,247 clones recovered from 63 duodenal biopsies obtained from healthy and celiac patients showed that R. mucilaginosa comprised [aprox.] 6% of the clones and was present in [aprox.] 65% of the biopsies, identifying it as a true colonizer of the duodenum [36]."

The million dollar question:

"The discovery of salivary microorganisms degrading dietary proteins in vitro prompts the question to what extent such microorganisms play a role in food processing in vivo. During mastication (chewing) foods are mixed with whole saliva helping to accelerate the break-down by digestive enzymes during the residency time in the oral cavity. Oral microorganisms in the swallowed food bolus may or may not survive and/or continue to exert proteolytic activities during or after gastric passage. Our in vitro data with R. aeria show that its enzymes are not abolished at acidic pH values, and are optimally active under more basic pH conditions. In vivo, this could mean that during gastric passage the enzymes will neither be active nor destroyed, and that enzymatic reactivation would occur upon transfer to the duodenum.

With regard to duodenal Rothia enzyme activity, it is relevant that R. mucilaginosa gains a foothold in the duodenum [36]. This offers the intriguing possibility that Rothia may colonize the duodenum and perform proteolytic activities locally in conjunction with mammalian- derived enzymes to degrade gluten."

The study here presented is a follow-up of one published in 2010 by the same group (2) in which they found that oral bacteria were capable of degrading completely immunogenic gliadin peptides. 

Further studies should help elucidate detailed information about the enzymes responsible for gluten degradation by these bacteria. 

This relationship might offer a way in which we are evolving and adapting to foods introduced in the neolithic: not only by changes in genes and gene expression (ie. AMY1), but also by establishing new symbiotic relationships with microorganisms. 

Zamakhchari M, Wei G, Dewhirst F, Lee J, Schuppan D, Oppenheim FG, & Helmerhorst EJ (2011). Identification of rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract. PloS one, 6 (9) PMID: 21957450