Dietary cystine restriction increases the proliferative capacity of the small intestine of mice

Over 88 million people are currently estimated to have adopted towards a vegan or vegetarian diet. Cysteine is a semi-essential amino acid, which availability is largely dependent on dietary intake of meat, eggs and whole grains. Vegan/vegetarian diets are therefore inherently low in cysteine concentrations. Sufficient uptake of cysteine is crucial, as it serves as substrate for protein synthesis and conversion to taurine and glutathione. In this study, we therefore investigate the effect of low dietary cystine, the oxidized derivative of cysteine, on intestinal epithelial layer function. Mice (8/group) received a high fat diet with normal or low cystine concentration for 2 weeks. We observed no changes in plasma methionine, cysteine, taurine or glutathione levels after 2 weeks. Stem cell markers as well as the proliferation marker Ki67 were increased upon cystine restriction in the small intestine. In line with this, gene set enrichment analysis indicated enrichment of Wnt signaling in the small intestine of mice on the low cystine diet, indicative of proliferative cells. Increased proliferation was absent in the colon. In the colon, dietary cystine restriction results in an increase in goblet cells, but no significant changes in the thickness of the mucus barrier or in its protective capacity. Also the microbiome was not changed upon dietary restriction. In conclusion, we show that cystine restriction for two weeks does not seem to induce any systemic effects. The increased proliferative capacity and number of goblet cells observed in the intestine may be the effect of starting epithelial damage or a reaction of the epithelium to start enlarging the absorptive capacity.


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Cysteine is a semi-essential amino acid, which is provided by dietary intake of meat, eggs and whole 44 grain. Cysteine can also be synthesized from the essential amino acid methionine via the 45 transsulfuration pathway (1,2). The majority of dietary cysteine is absorbed in the small intestine. Its 46 ileal uptake occurs by the cystine/glutamate exchange transporter, xCT (3). Unabsorbed cysteine 47 travels to the colon where it can be converted by sulfate-or sulfide-reducing bacteria to hydrogen 48 sulfide. Cystine is the oxidized form of cysteine. Compared to cysteine, cystine from food sources is 49 absorbed less efficient from the small intestine due to its lower digestibility (4). Intracellularly, 50 cystine is reduced to cysteine by the NADH-dependent enzyme cystine reductase (5).

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Next to protein synthesis, the three major fates of cysteine in the body are conversion to taurine and 52 glutathione and hydrogen sulfide. These three metabolites have differential effects on intestinal 53 function. Taurine is important for bile acid conjugation in the liver. Where in humans bile acids can 54 be conjugated with either glycine or taurine (3:1), mice conjugate 95% of bile acids with taurine (6).

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Specific taurine-conjugated bile acids like taurocholic acid and taurolithocholic acid have been 56 described to increase intestinal proliferation in intestinal cell lines (7,8). Additionally, taurine-57 conjugated bile acids increase the abundance of sulfite-reducing bacteria B. Wadsworthia , which 58 results in an increase in hydrogen sulfide production (9).

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Low hydrogen sulfide concentration has been shown to be beneficial for integrity of the mucus layer 60 (10), while excess hydrogen sulfide has been reported to cause breakdown of the mucus barrier and 61 induce DNA damage in enterocytes (10, 11).

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Lastly, glutathione (GSH) is an antioxidant which protects the intestine from oxidative stress and DNA 63 damage. The addition of glutathione to intestinal porcine enterocytes on a cysteine-deprived 64 medium restores proliferation and cell viability by replenishing the cysteine pool (12). Most of the 65 above mentioned studies mimick high cystine/cysteine intake, with concurrent high concentrations An increase in proliferation and stem cells can be indicative of intestinal damage and the necessity to 104 replenish the damaged cells (14,15). However, since we did not observe any significant changes in 105 body weight (Fig S1A), colon crypt length ( Fig S1B)  To unravel the underlying mechanism of the increased epithelial proliferation observed, we 118 investigated the effects of cystine restriction on the metabolic pathways involving cysteine. The 119 cystine-restricted diet did not cause a reduction in plasma cysteine levels (Fig 2A), suggesting that 120 either the dietary deficiency is compensated for by methionine-to-cysteine conversion, or by a 121 reduced production of taurine, glutathione and/or hydrogen sulfide. Plasma methionine levels were 122 also not changed, neither was the mRNA expression of cystathionine γ-lyase (CTH), which is 123 responsible for the last step in the conversion from methionine to cysteine (Fig 2A, 2B). This suggests 124 that the methionine-to-cysteine conversion is not increased. The cysteine-restricted diet did not 125 impact on glutathione production either, as both the plasma gluthathione concentration itself and 126 mRNA expression of enzymes involved in the conversion from cystine to glutathione (GSS, GCLC, 128 that there is no cysteine deficiency after 2 weeks on a low cystine diet, or that the low cystine 129 concentration (0.08%) still present in the low cys diet is sufficient.

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Cysteine restriction resulted in a trend towards decreased plasma taurine concentrations (Fig 2A).

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Methionine restriction has been shown to increase the ratio glycine to taurine conjugation of bile 132 acids in mice (17). We therefore hypothesized that a low cystine diet could influence the bile acid 133 conjugation ratio as well. Taurine and glycine conjugation of bile acids in gallbladder bile was not 134 changed ( Fig 2C), neither was total bile acid excretion into feces ( Fig 2D). Taken Fig 3A). This was confirmed by qRT-PCR for the Wnt 158 target genes Axin2, Ascl2, Cmyc, and CyclinD1 ( Fig 3B). As increased Wnt signaling is known to drive 159 proliferation and is essential for stem cell maintenance, the increased Wnt signaling seen in cysteine-160 restricted mice, most likely causes the observed effects on stem cells and proliferation. usage by bacteria. This hypothesis is supported by a study reporting that cystine supplementation 220 has a mucosal barrier enhancing effect (18). Whether this mechanism plays a role in this study needs 221 to be further investigated, but the fact that there are more goblet cells in the colon in the low cystine 222 group, even though the inner sterile layer is not thicker, might hint at a compensatory mechanism to 223 produce more mucus. Contradictory effects of cysteine, often in combination with methionine, on intestinal proliferation 225 have been reported in diverse disease states and animal models. Both sulfur amino acid (methionine 226 and cysteine) restriction and supplementation diets have been described to suppress intestinal 227 proliferation in weaned or neonatal pigs after a 7-day intervention (19,20). One study reports that 228 supplementation with both methionine and cysteine decreases expression of β-catenin in the small 229 intestine of weaned piglets, the downstream transcription factor in the Wnt pathway (19), after an 230 intervention of 7 days, which is in line with our findings that Wnt signaling is increased with cystine 231 restriction.

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Additionally, multiple studies examine the effect of cystine in cancer models, since cystine has been 233 reported to be essential for colorectal tumor growth. Cystine depletion has been described to reduce 234 tumor xenograft growth in a mouse model (21)  including its content was collected and centrifuged at 10 000 RCF for 10 min to collect the bile, which 259 was stored at -80 C.

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The colon was excised, mesenteric fat was removed, and the colon was opened longitudinally, washed 261 in PBS, and cut into 3 parts.