Can protein breaks provoke too much autophagy?

Reader Arash raised the interesting question whether the intermittent dietary protein restriction used in protein breaks could cause excessive autophagy.   This is important because too much autophagy can kill cells in certain settings, like oxygen deprivation in the brain (cerebral ischemia).

Excess autophagy apparently does not occur with intermittent protein restriction, because autophagy is self-limiting in this circumstance.  Restricting dietary protein, which is made of amino acids, deactivates mammalian target of rapamycin complex 1 (mTORC1), leading to protein degradation in cells by autophagy, which frees up amino acids, leading to reactivation of mTORC1 and consequent inhibition of autophagy again.

Laing explains:

[I]ncreased intracellular free amino acids produced during autophagic degradation can reactivate the mTORC1 signaling and thus downregulate autophagy, serving as a self-limiting feedback loop in autophagy regulation. ...

[I]t has been shown that mTORC1 can sense small increases in intracellular amino acids such as leucine, which leads to increased phosphorylation of both p70S6 kinase and its downstream target, ribosomal protein S6. Notably, such effects of amino acids on mTORC1 can be independent of stimulation by insulin or other growth factors, but are dependent on autophagic proteolysis. ...
A recent study by Yu et al. also showed that mTOR signaling is reactivated by prolonged starvation, which attenuates autophagy and restores lysosome homeostasis. This negative feedback loop is envisioned to be part of a homeostatic mechanism required to prevent prolonged or overactivation of autophagy.

Liang C. Negative regulation of autophagy. Cell Death Differ. 2010 December; 17(12): 1807.

Also, Yu finds "mTOR signaling is inhibited during autophagy initiation, but reactivated with prolonged starvation. mTOR reactivation is autophagy-dependent, and requires the degradation of autolysosomal products."

Yu L, et al. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature. 2010;465:942.

See also: Avruch J, et al. Amino acid regulation of TOR complex 1. Am J Physiol Endocrinol Metab. 2009;296.

Why take protein breaks?

This blog (and this) presents an idea that, based on current research, appears likely true and may save your life:

Taking intermittent "protein breaks," when you eat very little or no protein for 2-4 consecutive days while eating plenty of carbohydrate, can slow aging and prevent, delay, or reverse many diseases including obesity, type 2 diabetes, autoimmune disorders, and brain diseases such as Alzheimer's and Parkinson's.

If you take protein breaks, you must follow each by a period of eating adequate amounts of protein.

This hypothesis awaits confirmation by randomized human clinical trials, but it appears consistent with prior studies in humans, other animals, and living cells.

Because no clinical trial has yet been done to establish the efficacy of protein breaks in humans, I cannot recommend them.  But I recommend you consider them with your doctor.

Alzheimer’s at EMBO 2013: dietary treatment and prevention through autophagy

Here's a PDF of my poster presented at the EMBO autophagy conference in May 2013.  And here's the abstract.

The poster's text:

An autophagic role in Alzheimer's disease for intermittent dietary periods of very low-protein, high-carbohydrate intake

Hypothesis: Intermittent periods of very low-protein, high-carbohydrate dietary intake may enhance autolysosomal proteolysis in Alzheimer's disease (AD) by increasing activity of transcription factor EB (TFEB).

Background: AD is characterized by 1) activation of neuronal autophagy with defective autolysosomal degradation,^[1] and 2) neuronal insulin resistance, characterized by increased amyloid-β (Aβ) production in autophagosomes and reduced neuronal internalization of extracellular Aβ oligomers.^[2] 

Translocation of transcription factor EB (TFEB) from cytosol to nucleus increases transcription of 291 genes and thereby induces autophagy,^[3] lysosomal biogenesis, acidification, and proteolysis.^[4]

Phosphorylation of TFEB by mammalian target of rapamycin complex 1 (mTORC1) and by glycogen synthase kinase 3 (GSK3)^[5] inhibits TFEB nuclear translocation.

GSK3 inhibition in transgenic AD mice increases acidification of lysosomes, reduces Aβ deposits, and ameliorates cognitive deficits.^[6]

Why very low protein intake?  mTORC1 phosphorylation of TFEB is inhibited by amino acid starvation, even in the presence of strong insulin signaling.^[7]  Very low protein intake, combined with GSK3 inhibition, is therefore expected to promote TFEB nuclear translocation.

Why high carbohydrate intake?  High carbohydrate intake stimulates secretion of insulin, which inhibits GSK3^[8] and presumably therefore reduces GSK3's phosphorylation of TFEB.  Combined with mTORC1 inhibition, enhanced insulin signaling should thereby promote TFEB nuclear translocation.

This hypothesis awaits testing, e.g., in a transgenic AD mouse model.

---

^[1] Nixon RA, Yang DS. Autophagy failure in Alzheimer's disease—locating the primary defect. Neurobiology of Disease (2011) 43(1): 38-45.

^[2] Talbot K, et al. Demonstrated brain insulin resistance in Alzheimer’s disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline. J Clin Invest. (2012) 122(4): 1316–1338.

^[3] Settembre C, et al. TFEB Links Autophagy to Lysosomal Biogenesis. Science (2011) 332(6036): 1429-1433.

^[4] Sardiello M, et al. A Gene Network Regulating Lysosomal Biogenesis and Function. Science (2009) 325(5939): 473-477.

^[5] Parr C, et al. GSK3 inhibition promotes lysosomal biogenesis and the autophagic degradation of the Amyloid-β Precursor Protein. Mol. Cell. Biol. (2012) 32(21): 4410-4418.

^[6] Avrahami L, et al. Inhibition of GSK-3 Ameliorates beta-Amyloid(A-beta) Pathology and Restores Lysosomal Acidification and mTOR Activity in the Alzheimer's Disease Mouse Model. In vivo and In vitro Studies.  J Biol Chem (2012) Nov 15.

^[7] Settembre C, et al.  A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. The EMBO Journal (2012) 31, 1095-1108.

^[8] Collino M, et al. Insulin Reduces Cerebral Ischemia/Reperfusion Injury in the Hippocampus of Diabetic Rats. A Role for Glycogen Synthase Kinase-3β.  Diabetes (2009) 58(1): 235-242.

Protein restriction cycles improve behavior and reduce IGF-1 and phosphorylated Tau in Alzheimer's mice

A recent University of Southern California study showed great benefit in cycling intake of dietary protein -- well, not all protein, but essential amino acids -- in mice with Alzheimer's disease.

The protein restriction cycles improved memory and reduced brain levels of phosphorylated tau in the mice, but did not affect brain levels of β amyloid (Aβ) plaques.

The protein cycles lasted four months and consisted of alternating weeks of a normal diet and a protein-restricted (PR) diet.

The normal diet contained 25% protein, 17% fat, and 58% carbohydrate.

The PR diet lacked nine essential amino acids (EAA) – that is, amino acids the body cannot make: isoleucine, leucine, lysine, methionine, phenyalanine, threonine, tryptophan, valine, and arginine. Fat and carbohydrate contents were presumably the same as in the normal diet.

Interestingly, the researchers supplemented the PR diet by adding more of the remaining 11 amino acids, mainly the nonessential amino acids (NEAA), to make the diet's nitrogen content the same as in the normal control diet. As a result, the PR diet contained about twice the amount of NEAA as did the normal diet.

The mice in this study were triple transgenic Alzheimer's (3xTg-AD) mice, which overexpress two human genes (presenilin-1 and amyloid precursor protein (APP)) having mutations linked to Alzheimer's disease, and one (tau) linked to frontotemporal dementia. These mutations result in development of both Aβ plaques and phosphorylated tau tangles in the brain, as well as age-dependent Alzheimer-like cognitive impairment.

Here is the article's abstract:

In laboratory animals, calorie restriction (CR) protects against aging, oxidative stress, and neurodegenerative pathologies. Reduced levels of growth hormone and IGF-1, which mediate some of the protective effects of CR, can also extend longevity and/or protect against age-related diseases in rodents and humans. However, severely restricted diets are difficult to maintain and are associated with chronically low weight and other major side effects. Here we show that 4 months of periodic protein restriction cycles (PRCs) with supplementation of nonessential amino acids in mice already displaying significant cognitive impairment and Alzheimer's disease (AD)-like pathology reduced circulating IGF-1 levels by 30-70% and caused an 8-fold increase in IGFBP-1. Whereas PRCs did not affect the levels of β amyloid (Aβ), they decreased tau phosphorylation in the hippocampus and alleviated the age-dependent impairment in cognitive performance. These results indicate that periodic protein restriction cycles without CR can promote changes in circulating growth factors and tau phosphorylation associated with protection against age-related neuropathologies.

Study authors included USC's Valter Longo and Pinchas Cohen, and Luigi Fontana of Washington University in St. Louis.

Parrella E, et al. Protein restriction cycles reduce IGF-1 and phosphorylated Tau, and improve behavioral performance in an Alzheimer's disease mouse model.
Aging Cell. 2013 Apr;12(2):257-68. doi: 10.1111/acel.12049.

An autophagic role in Alzheimer's disease for intermittent dietary periods of very low-protein, high-carbohydrate intake

Here's the text of an abstract I'll be presenting at the European Molecular Biology Organization (EMBO) autophagy conference in Norway in May 2013:

….

Occasional periods of very low-protein, high-carbohydrate dietary intake may enhance lysosomal proteolysis in Alzheimer's disease (AD) by increasing activity of transcription factor EB (TFEB) via inhibition of glycogen synthase kinase 3 (GSK3).

AD is characterized by 1) activation of neuronal autophagy with defective autolysosomal degradation, and 2) neuronal insulin resistance, characterized by increased amyloid-β (Aβ) production in autophagosomes and reduced neuronal internalization of extracellular Aβ oligomers.

Suitable AD therapies may therefore aim to reduce neuronal insulin resistance and increase activity of TFEB, a master gene regulator of lysosomal biogenesis. Upon cellular starvation and in response to inhibition of mammalian target of rapamycin (mTOR), TFEB translocates from the cytosol to the nucleus, whereupon it increases transcription of 291 genes, including many involved in autophagy. At least 20 of these genes participate in lysosomal biogenesis, acidification, and proteolysis.

The mTOR inhibitor rapamycin apparently cannot induce lysosomal biogenesis by TFEB, as rapamycin does not blunt mTORC1's nutrient-induced phosphorylation of TFEB at Serine 142, which keeps TFEB localized in the cytosol.

But GSK3 also phosphorylates TFEB at S142, and GSK3 inhibition results in translocation of unphosphorylated TFEB into the nucleus, increasing transcription of lysosomal genes and degradation of Aβ.GSK3 inhibition in AD may further be useful because GSK3 hyperphosphorylates tau, increases Aβ production, and impairs memory.

As recent proof of concept, treatment of mice expressing mutated amyloid precursor protein (APP) and presenilin-1 (PS1) with the selective GSK3 inhibitor L803-mts increased acidification of lysosomes, reduced Aβ deposits, and ameliorated cognitive deficits.

Insulin apparently exerts opposing actions with respect to TFEB, both (a) activating mTORand thereby decreasing TFEB nuclear localization, and (b) inhibiting GSK3and thereby increasing TFEB nuclear localization. If the effect of insulin's mTOR-mediated decrease in nuclear localization of TFEB could be lessened, while maintaining the effect of insulin's GSK3 inhibition-mediated increase in that localization, then insulin could be harnessed to augment lysosomal biogenesis.

Settembre et al. found that the constitutive activation of growth factor (e.g., insulin) inputs to mTORC1 occurring in TSC2 -/- cells could not suppress nuclear translocation of TFEB in response to amino acid starvation. This suggests that during intermittent periods of very low dietary protein intake, elevating serum insulin concentrations to inhibit GSK3, such as by a high dietary carbohydrate intake, may promote nuclear localization of TFEB and its consequent stimulation of lysosomal transcription. In other words, during autophagy induced by amino acid deprivation, high insulin signaling may induce more lysosomal biogenesis than does low insulin signaling, such as during starvation.

Moreover, there is evidence suggesting that intermittent dietary protein restriction may reduce insulin resistance in the AD brain. For example, elevated amino acid levels in humans induce insulin resistance in skeletal muscle via activation of mTOR and ribosomal protein S6 kinase 1 (S6K1). And severe protein restriction both decreases insulin requirements in type 1 diabetics and decreases fasting hepatic glucose output and basal insulin levels in normal subjects.

A dietary regime of intermittent very low-protein and high-carbohydrate intake may thus be effective in preventing and treating AD.

A study implementing this strategy in a transgenic AD mouse model is proposed.

(Citations omitted.)