Cultured skin fibroblasts from patients with tuberous sclerosis were analyzed as to their sensitivities to proline, glutamic acid and arginine, and their analogues by means of a cell growth study. A difference was observed between tuberous sclerosis and normal cells when proline was added. Skin fibroblasts derived from normal-appearing skin of tuberous sclerosis patients exhibited sensitivity to proline. Skin fibroblasts derived from tumorous skin of tuberous sclerosis patients exhibited resistance to proline in the cell growth study and was found to be rather sensitive to proline in a colony formation study. This aberrant sensitivity to proline of tuberous sclerosis cells may indicate abnormal proline metabolism in tuberous sclerosis patients.

Thank you very much Heshmat. Can you give me some indication of articles on the subject?

Maybe even of proline and hyperfunction of mTOR pathway.

Article Proline Metabolism and Microenvironmental Stress

...proline degradation may be playing a role under conditions when mTOR

is inhibited.

Article Regulation and Function of Proline Oxidase Under Nutrient Stress

I am not aware of any recent connection of proline metabolism to tuberous sclerosis complex (TSC).  In the 80's, the genes for TSC were unknown, as was the link to mTOR.  Therefore, the relevance to the disease state of the cells studied in those papers is unclear because it was essentially impossible to determine at the time. 

Today, one would want to determine whether the cells being studied had measurably increased mTOR activity (e.g., phospho-S6) and whether they had loss of heterozygosity or remained heterozygous for a TSC1 or TSC2 mutation.  One would also want to know more about the phenotype of the cells and to ask, for example, if fibroblasts are a relevant cell type and whether culture conditions may influence assay results.  Given the lack of clarity underlying the experiments and absence of any similar data from other laboratories over a 30-year timespan, I would hesitate to use those data as a starting point for further research.

However, many data suggest that there is a particular interaction between the proline metabolism and the mTOR pathway

Growth control via TOR kinase signaling, an intracellular sensor of amino acid and energy availability, with crosstalk potential to proline metabolism.

http://www.ncbi.nlm.nih.gov/pubmed/18651095

http://ajpcell.physiology.org/content/ajpcell/298/5/C982.full.pdf

http://cancerres.aacrjournals.org/content/72/14/3677.full.pdf

Article Proline dehydrogenase is essential for proline protection ag...

Article L-Proline induces differentiation of ES cells: a novel role ...

The mTORC1/S6K1 Pathway Regulates Glutamine Metabolism through the eIF4B-Dependent Control of c-Myc Translation.

Alfredo Csibi, Gina Lee, Sang-Oh Yoon, Haoxuan Tong, Didem Ilter, Ilaria Elia, Sarah-Maria Fendt, Thomas M Roberts, John Blenis

ABSTRACT: Growth-promoting signaling molecules, including the mammalian target of rapamycin complex 1 (mTORC1), drive the metabolic reprogramming of cancer cells required to support their biosynthetic needs for rapid growth and proliferation [1]. Glutamine is catabolyzed to α-ketoglutarate (αKG), a tricarboxylic acid (TCA) cycle intermediate, through two deamination reactions, the first requiring glutaminase (GLS) to generate glutamate and the second occurring via glutamate dehydrogenase (GDH) or transaminases [2]. Activation of the mTORC1 pathway has been shown previously to promote the anaplerotic entry of glutamine to the TCA cycle via GDH. Moreover, mTORC1 activation also stimulates the uptake of glutamine, but the mechanism is unknown [3]. It is generally thought that rates of glutamine utilization are limited by mitochondrial uptake via GLS, suggesting that, in addition to GDH, mTORC1 could regulate GLS. Here we demonstrate that mTORC1 positively regulates GLS and glutamine flux through this enzyme. We show that mTORC1 controls GLS levels through the S6K1-dependent regulation of c-Myc (Myc). Molecularly, S6K1 enhances Myc translation efficiency by modulating the phosphorylation of eukaryotic initiation factor eIF4B, which is critical to unwind its structured 5' untranslated region (5'UTR). Finally, our data show that the pharmacological inhibition of GLS is a promising target in pancreatic cancers expressing low levels of PTEN.

Article The mTORC1/S6K1 Pathway Regulates Glutamine Metabolism throu...

Article Synthetic lethality of combined glutaminase and Hsp90 inhibi...

Apoptotic effects of high-dose rapamycin occur in S-phase of the cell cycle

Mahesh Saqcena et al. 

Cell cycle (Georgetown, Tex.) (Impact Factor: 5.01). 04/2015; 

ABSTRACT Mutations in genes encoding regulators of mTOR, the mammalian target of rapamycin, commonly provide survival signals in cancer cells. Rapamycin and analogs of rapamycin have been used with limited success in clinical trials to target mTOR-dependent survival signals in a variety of human cancers. Suppression of mTOR predominantly causes G1 cell cycle arrest, which likely contributes to the ineffectiveness of rapamycin-based therapeutic strategies. While rapamycin causes the accumulation of cells in G1, its effect in other cell cycle phases remains largely unexplored. We report here that when synchronized MDA-MB-231 breast cancer cells are allowed to progress into S-phase from G1, rapamycin activates the apoptotic machinery with a concomitant increase in cell death. In Calu-1 lung cancer cells, rapamycin induced a feedback increase in Akt phosphorylation at Ser473 in S-phase that mitigated rapamycin-induced apoptosis. However, sensitivity to rapamycin in S-phase could be reestablished if Akt phosphorylation was suppressed. We recently reported that glutamine (Gln) deprivation causes K-Ras mutant cancer cells to aberrantly arrest primarily in S-phase. Consistent with observed sensitivity of S-phase cells to rapamycin, interfering with Gln utilization sensitized both MDA-MB-231 and Calu-1 K-Ras mutant cancer cells to the apoptotic effect of rapamycin. Importantly, rapamycin induced substantially higher levels of cell death upon Gln depletion than that observed in cancer cells that were allowed to progress through S-phase after being synchronized in G1. We postulate that exploiting metabolic vulnerabilities in cancer cells such as S-phase arrest observed with K-Ras-driven cancer cells deprived of Gln, could be of great therapeutic potential.

Article Apoptotic effects of high-dose rapamycin occur in S-phase of...

Introduction: Tuberous sclerosis complex (TSC) is a genetic disease resulting from mutation in TSC1 or TSC2 and subsequent hyperactivation of mammalian Target of Rapamycin (mTOR). Common TSC features include brain lesions, such as cortical tubers and subependymal giant cell astrocytomas (SEGAs). However, the current treatment with mTOR inhibitors has critical limitations. We aimed to identify new targets for TSC pharmacotherapy.

Results: The results of our shRNA screen point to glutamate-cysteine ligase catalytic subunit (GCLC), a key enzyme

in glutathione synthesis, as a contributor to TSC-related phenotype. GCLC inhibition increased cellular stress and reduced mTOR hyperactivity in TSC2-depleted neurons and SEGA-derived cells. Moreover, patients’ brain tubers showed elevated GCLC and stress markers expression. Finally, GCLC inhibition led to growth arrest and death of SEGA-derived cells.

Conclusions: We describe GCLC as a part of redox adaptation in TSC, needed for overgrowth and survival of mutant cells, and provide a potential novel target for SEGA treatment.

Keywords: Tuberous Sclerosis Complex, Glutamate-cysteine ligase, Cellular stress, Brain tumors, Cell death

Article Tuberous sclerosis complex neuropathology requires glutamate...

.

Article Prolidase-proline dehydrogenase/proline oxidase-collagen bio...

Hepatic mTORC1 controls locomotor activity, body temperature, and lipid metabolism through FGF21

Marion Cornua, Wolfgang Oppligera, Verena Alberta, Aaron M. Robitaillea, Francesca Trapanib, Luca Quagliatab, Tobias Fuhrerc, Uwe Sauerc, Luigi Terraccianob, and Michael N. Halla.

1592–11599 | PNAS | August 12, 2014 | vol. 111 | no. 32

Finally, glutaminolysis both promotes mTORC1 signaling (55) and is activated by mTORC1 (56). mTORC1 activates glutaminolysis throughrepression of the glutamate dehydrogenase inhibitor SIRT4.

Our results demonstratethat hepatic mTORC1 activation, due to Tsc1knockout specifically in the liver, causes glutamine depletion and thereby PGC-1α–dependent FGF21 expression.

mTORC1 promotes glutaminolysis and consequently mTORC1 hyperactivation in Tsc2-deficient cells leads to glutamine depletion.

The i.p. injection of rapamycin restored normal glutamine levels in L-Tsc1KO mice.

Article Hepatic mTORC1 controls locomotor activity, body temperature...

Glutamine, the most abundant free amino acid, is an

important substrate for the proline synthetic pathway,

but proline is also stored in the ECM as collagen which

can be a reservoir or ‘dump’ for proline.

Article Proline metabolism and cancer: Emerging links to glutamine a...

Glutamate acts as a nitrogen donor for the transamination involved in the production of 'dispensable amino acids' — alanine, aspartate and serine — through the actions of glutamate–oxaloacetate transaminase (GOT), glutamate–pyruvate transaminase (GPT) and phosphoserine aminotransferase 1 (PSAT1), respectively. Glutamine can also act as a nitrogen donor for asparagine through asparagine synthetase (ASNS). In a reaction independent of transamination, proline can be synthesized by conversion of glutamate to pyrroline-5-carboxylate (P5C) by pyrroline-5-carboxylate synthase (P5CS; also known as ALDH18A1) and subsequently to proline by pyrroline-5-carboxylate reductase 1 (PYCR1) and PYCR2. Glutamine also contributes to the tripeptide glutathione (composed of glutamate, cysteine and glycine), which neutralizes the reactive oxygen species (ROS), including H2O2 (Ref. 110). The first step in glutathione synthesis is the condensation of glutamate and cysteine through glutamate–cysteine ligase (GCL; not shown in the figure). Glutamine input contributes directly to the availability of cysteine and glycine for production of glutathione. Glutamate can be exchanged for cystine (which is quickly reduced to cysteine inside the cell) through the xCT antiporter (a heterodimer of SLC7A11 and SCL3A2), which has been shown to be important in various cancers and has been considered as a drug target18, 212. Glycine is next added by glutathione synthetase (GSS; not shown in the figure). Additionally, glutamate can contribute to glycine through transamination by PSAT1 into phosphoserine (pSer) and α-ketoglutarate (α-KG) and subsequent conversion to glycine through serine hydroxymethyltransferase (SHMT; not shown in the figure) as part of the one-carbon metabolism pathway, which has been shown in numerous studies to be crucial in cancer metabolism and is also reviewed in this Focus issue by Vousden139, 140, 213. GLS, kidney-type glutaminase; GLS2, liver-type glutaminase; GLUD, glutamate dehydrogenase; OAA, oxaloacetate. (Link 1)

Depletion of p62 in Tsc2-null cells decreased intracellular glutamine, glutamate, and glutathione.. (Link 2)

http://www.nature.com/nrc/journal/v16/n10/full/nrc.2016.71.html

Article p62/SQSTM1 Cooperates with Hyperactive mTORC1 to Regulate Gl...

Maurizio:

A good deal of the attention since Tanaka's pioneer research on tuberous sclerosis complex (TSC) has of late shifted to leveraging underlying molecular pathways, especially the proline-rich Akt and mTOR pathways [Oshiro 2007], and given that TSC appears to result from the constitutive activation of mTOR (and TSC itself being a negative regulator of mTORC1 signaling), mTOR inhibitors have found their way into the TSC tretament setting [Wataya-Kaneda 2015] [Inoki 2009] [Orlova 2010], with some clinical success from the mTOR inhibitor everolimus (Afinitor), especially in the connection with autism spectrum disorder (ASD) [Davis 2015] [Tye 2016]. This is being pursued aggressively in several innovative clinical trials, with even topical administration of mTOR inhibitors like everolimus showing  some considerable promise.

References

[Davis 2015] Davis PE, Peters JM,Kreuger DA, Sahin M. Tuberous Sclerosis: A New Frontier in Targeted Treatment of Autism. Neurotherapeutics 2015; 12(3):572-83.

[Inoki 2009] Inoki K, Guan KL. Tuberous sclerosis complex, implication from a rare genetic disease to common cancer treatment. Hum Mol Genet 2009 Apr 15; 18(R1):R94-100.

[Orlova 2010] Orlova KA, Crino PB. The tuberous sclerosis complex. Ann N Y Acad Sci 2010; 1184:87-105.

[Oshiro 2007] Oshiro N, Takahashi R, Yoshino K, et al. The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1. J Biol Chem 2007 Jul 13; 282(28):20329-39.

Tye C, Varcin K, Bolton P, Jeste SS. Early developmental pathways to autism spectrum disorder in tuberous sclerosis complex. Advances in Autism 2016; 2(2):84-93.

[Wataya-Kaneda 2015] Wataya-Kaneda M. Mammalian target of rapamycin and tuberous sclerosis complex. J Dermatol Sci. 2015 Aug;79(2):93-100.]

Constantine

We also measured 20 amino acids linked to the TCA cycle (Fig. 3) and found their concentrations were changed by everolimus, with some decreased (glycine, proline) and others were elevated (serine, alanine, aspartic acid, threonine, cysteine, glutamic acid, glutamine, and methionine). 

https://link.springer.com/content/pdf/10.1007%2Fs11306-017-1236-5.pdf

http://www.cell.com/cell-metabolism/fulltext/S1550-4131(16)30424-7

Article Proline metabolism supports metastasis formation and could b...

Article p62/SQSTM1 Cooperates with Hyperactive mTORC1 to Regulate Gl...

Article Proline Metabolism in Cell Regulation and Cancer Biology: Re...

Brain Dev. 1987;9(1):37-42.

Tuberous sclerosis: aberrant metabolism of ornithine, proline and glutamate in cultured fibroblasts.

Tanaka H, Nakazawa K, Arima M, Hayashi A.

Abstract

To investigate aberrant metabolism of proline (Pro) and its precursors in tuberous sclerosis (TS), 6, 7 and 5 strains of control, TS (normal skin) and TS (tumor) fibroblasts, respectively, were cultured in Eagle's MEM containing dialyzed fetal bovine serum with or without 0.1 mM ornithine (Orn). Ornithine aminotransferase (OAT) activity was decreased in TS, especially in TS (tumor) after mild sonication treatment. The yield of the OAT protein was inhibited in TS (tumor) when cultured without Orn. Free glutamate (Glu) in the medium was significantly increased in TS (tumor). Free proline (Pro) in cells was significantly decreased in TS (tumor) when cultured with Orn, but protein-bound Pro was not. The relative concentration of free Glu to glutamine (Gln) in the medium and that of free Glu to Pro in cells cultured with Orn were increased in TS (tumor). These results suggest that the requirement for Orn, increased turnover of Pro to Glu and increased elimination of Glu into the medium occur in TS (tumor). Aberrant regulation or turnover of Pro and Glu metabolism may occur in TS, especially in tumor cells.

Neuropsychopharmacology. 2017 Dec 5. doi: 10.1038/npp.2017.295.

mGluR5 Modulation of Behavioral and Epileptic Phenotypes in a Mouse Model of Tuberous Sclerosis Complex.

Kelly E1,2, Schaeffer SM1, Dhamne SC1, Lipton JO1,3, Lindemann L4, Honer M5, Jaeschke G6, Super CE1, Lammers SH1, Modi ME1, Silverman JL7, Dreier JR8, Kwiatkowski DJ8, Rotenberg A1, Sahin M1.

Abstract

Drugs targeting metabotropic glutamate receptor 5 (mGluR5) have therapeutic potential in autism spectrum disorders (ASD), including Tuberous Sclerosis Complex (TSC). The question whether inhibition or potentiation of mGluR5 could be beneficial depends, among other factors, on the specific indication. To facilitate the development of mGluR5 treatment strategies, we tested the therapeutic utility of mGluR5 negative and positive allosteric modulators (an mGluR5 NAM and PAM) for TSC, using a mutant mouse model with neuronal loss of Tsc2 that demonstrates disease-related phenotypes including behavioral symptoms of ASD and epilepsy. This model uniquely enables the in vivo characterization and rescue of the electrographic seizures associated with TSC. We demonstrate that inhibition of mGluR5 corrects hyperactivity, seizures, and elevated de novo synaptic protein synthesis. Conversely, positive allosteric modulation of mGluR5 results in the exacerbation of hyperactivity and epileptic phenotypes. The data suggest a meaningful therapeutic potential for mGluR5 NAM's in TSC, which warrants clinical exploration and the continued development of mGluR5 therapies. Neuropsychopharmacology accepted article preview online, 05 December 2017.

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The combination of these two articles (one of which is very ancient from which began this small collection of notes and the second most recent of which I read only the abstract but I hope to read soon the entire article by Kelly) does not necessarily mean that the positive effect on seizures obtained with inhibition of mGluR5 is trivially related to an excess of Glutamate. The GluRs are in fact involved in multiple metabolic and signaling pathway (including also mTOR pathway, progression of tumors with various histotypes and the presence of receptors in various extracerebral organs etc.). For now I would just like to point out another robust signal of the correlation between proline metabolism and tuberous sclerosis.

Article Metabolomic studies identify changes in transmethylation and...

Tuberous sclerosis complex (TSC) is an autosomal dominant neurodevelopmental disorder and the quintessential disorder of mTORC1 dysregulation. Loss of either causative gene, TSC1 or TSC2, leads to constitutive mTORC1 kinase activation and a pathologically anabolic state of macromolecular biosynthesis. Little is known about the organ-specific metabolic reprogramming that occurs in TSC-affected organs. Using a mouse model of TSC in which Tsc2 is disrupted in radial glial precursors and their neuronal and glial descendants, we performed an unbiased metabolomic analysis of hippocampi to identify Tsc2-dependent metabolic changes. Significant metabolic reprogramming was found in well-established pathways associated with mTORC1 activation, including redox homeostasis, glutamine/TCA cycle, pentose and nucleotide metabolism. Changes in two novel pathways were identified: transmethylation and polyamine metabolism. Changes in transmethylation included reduced methionine, cystathionine, S-adenosylmethionine (SAM - the major methyl donor), reduced SAM/SAH ratio (cellular methylation potential), and elevated betaine, an alternative methyl donor. These changes were associated with alterations in SAM-dependent methylation pathways and expression of the enzymes methionine adenosyltransferase 2A (MAT2A) and cystathionine beta synthase (CBS). We also found increased levels of the polyamine putrescine due to increased activity of ornithine decarboxylase, the rate-determining enzyme in polyamine synthesis. Treatment of Tsc2+/- mice with the ornithine decarboxylase (ODC) inhibitor 2-difluoromethylornithine (DFMO), to reduce putrescine synthesis dose-dependently reduced hippocampal astrogliosis. These data establish roles for SAM-dependent methylation reactions and polyamine metabolism in TSC neuropathology. Importantly, both pathways are amenable to nutritional or pharmacologic therapy.