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

Advanced cancer and the ketogenic diet

A new pilot trial about cancer-ketogenic diet has recently been published (1). This german group evaluated specifically the feasibility of a KD and its influence on the quality of life of patients with advanced metastatic tumors. The full text is free, so anyone can go and check the details and methodology used. The nutritional intervention was basically an ad libitum KD (<70g/CHO/day), plus extra omega-3 fatty acids. 

Dietary guidelines for the patients were (Table 2):

1. Avoid all types of bread, cake, processed snacks, sweets, potatoes, pasta,
   rice, polenta, vegetables rich in starch (corn, beans, peas) and cereals.
2. Be aware of hidden sources of CHO in sugar sweetened drinks, candy,
   chewing gum with sugar, milk and milk products, lunch meat and some
   cheeses as well as in most “low fat” products.
3. Fruits are rich in CHO, therefore always calculate the amount and select
   those which are low in CHO.
4. Vegetables are often rich in CHO - but mainly in dietary fiber, therefore
   calculate the usable CHO only.
5. If possible, prefer cold-water fish and meat from grazing cattle as protein
   sources, because of their preferable fatty acid pattern.
6. Vegetables and the few fruits allowed should be grown organic
7. As nibbles, select oil-rich nuts (walnuts, brazil nuts, macadamia nuts) and
   seeds (sunflower), and only occasionally chocolate with very high cacao
   content (min. 85%).

This guidelines look very paleoish to me (whatever that means). Some things I found interesting are points 5 and 6. Not seen emphasized commonly. Additionally, patients were told to drink two liquid meals as snacks. Components of this shake were provided to the patients and included: 250ml of highly fermented yogurt-drink, 8ml vegetable oil mixture and 10g of protein preparation. Ingredients of the components can be seen in Table 3. 

Because of problems with compliance to the diet, from 16 initial participants, only 5 remained until the end of the study (31%). Two patients dropped out during the first week, one because of inability to adhere to the diet and the other because of personal problems. Two patients died from their malignant disease during the study, one patient dropped out because he suffered excessive weight loss and weakness, one patient quit because he felt he wasnt able to stick to the dietary guidelines, one because resuming chemotherapy and four due to progress of their advanced cancer situation. The compliance problem is common. Adopting a ketogenic diet involves a lifestyle change. Something even advanced cancer patients can't do. This reminds me of a study in which some cancer patients wouldnt adopt a KD because that meant "giving up the candies and ice cream", despite the fact that it could improve their condition. To complicate things further, the acceptance of the diet varied greatly. One patient said that after 3 days on the diet it was not feasible at all and stopped the diet. Two patients rate feasibility as "very good", seven patients rate it "good", three "moderate" and one "poor". This was after 2 weeks of dieting so included the 16 initial participants.

Quality of life was measured by the EORTC QLQ-C30 questionnaire. Global scores remained relatively stable during the evaluation time. Physical and role functioning worsened slightly over time and constipation was reported by most patients. Because of the advanced cancer stage of the patients, fatigue, pain or dyspnoea increased over time. Nevertheless, emotional functioning increased slightly and insomina improved. 

Of those who completed the whole 12 weeks of dieting, 60% reached a stable ketonuria, predominantly being 1.5-4.0mM. Among blood parameters, only some patients had available data. Overall, CRP levels increased slighlty over time, considering the initial values were high. Two patients initially had elevated glucose, which returned to normal. In other patients, cholesterol levels were "normalized" (meant by reduced to conventionally accepted levels), as well as triglycerides in one patient and ALT in other patient. Total leukocyte count significantly increased during the intervention (even though one patient with initial low leukocyte counts showed a further reduction). 

Patients lost an average of 2kg. Progress of the disease occured in 5 patients who then discontinued the diet, while 5 patients who adhered to the diet had stable disease progression.

Overall, the percentage of days in ketosis (>0.5mmol/l) was not correlated with the results of the study. We can see in table 4 that for example, patient 6 reached 97% of days in ketosis, but because of impaired food intake only completed 6 weeks and showed progress in the disease. On the other hand, patient 16 reached 100% days in ketosis, completed the trial and showed no progress in the disease. Both patients 5 and 11 only reached 25% of days in ketosis, but completed the trial and maintained their condition. 

The limitations of this study were:

- Patients had advanced stage cancer. While a KD might help preventing and/or treating some cancers, there is no much left to do when the disease is too severe.

- Most patients werent from the author's hospital. Blood samples and laboratory parameters had to be provided by their family doctors or local oncologists.

- Short sample and short intervention time. 

Being fair, at the time the study was done (2007) guidelines to apply a KD for cancer treatment were scarce. The only premise was that reducing carbohydrates (hence sugar and cancer's fuel) would reduce progression of tumors. Since then, there is more information available which suggest how to implement the KD for these patients. Overall, evidence suggest that the diet should be not only ketogenic, but calorie restricted. This is for achieving low blood glucose levels and increased KB. In the study reviewed in this post, calories were ad libitum and with a carbohydrate intake limited to 70g/day. Glucose should be ideally around 55-65mg/dl and KB 4-7mM. Checking the study data, most patients had much higer BG (mean 93) and only mild ketosis. Therapeutic fasting is another valuable tool, but harder to comply with. Another factor to take into account is the cancer phenotype. A restricted KD should be more efficient in predominantely glucose-consuming tumors, which can be assessed using some phenotypic markers. Serum LDH levels, for instance, have been shown to be correlated with activation of HIF related genes (2), which include glycolytic enzymes (3). Or using the more conventional FDG-PET. Finally, utilization of gluconeogenesis and glycolysis inhibitors (ie. 2-Deoxyglucose or metformin) with the KD has also been proposed (4,5).

Recent evidence suggests that this metabolic therapy is promising. One case report (6) has shown a rapid regression of glioblastoma multiforme in an old patient using the guidelines proposed by Seyfried et al (7). This patient started the metabolic therapy with a water-only fast, switching then to a restricted KD which delivered 600kcal/day for 14 days. Dexamethasone was also eliminated (because high dosage steroid medication increases gluconeogenesis and blood glucose levels, while enhancing apoptosis resistance in tumor cells). Because of development of mild hiperuricemia, the KD was changed for a non-ketogenic calorie-restricted diet which also delivered 600kcal/day. Aside from the complete regression in such a short time (2-2.5 months), the most surprising finding in my opinion was the recurrence of the tumor after discontinuing the metabolic therapy, which strongly suggests that the therapy itself was the most infulential factor in cancer regression.

It seems that controlling blood glucose levels is the more important part of the metabolic therapy. Achieving blood glucose levels of 55-65mg/dl and 4-7mM of ketone bodies has been termed as "the zone of metabolic management":

This resembles the results from the study on the GB patient:

Although target blood glucose levels were not achieved, the reduction observed was sufficient for controlling disease progression. The other difference was the method of detection of ketosis, urinary ketones, which doesnt always correlate to blood ketone levels. 

Schmidt M, Pfetzer N, Schwab M, Strauss I, & Kammerer U (2011). Effects of a ketogenic diet on the quality of life in 16 patients with advanced cancer: A pilot trial. Nutrition & metabolism, 8 (1) PMID: 21794124