Methionine restriction fundamentally supports health by tightening epithelial barriers

Abstract

Dietary methionine restriction (MR) has been found to affect one of the most primary tissue-level functions of an organism: the efficiency with which the epithelial linings of major organs separate the fluid compartments that they border. This process, epithelial barrier function, is basic for proper function of all organs, including the lung, liver, gastrointestinal tract, reproductive tract, blood-brain barrier, and kidney. Specifically, MR has been found to modify the protein composition of tight junctional complexes surrounding individual epithelial cells in a manner that renders the complexes less leaky. This has been observed in both a renal epithelial cell culture model and in gastrointestinal tissue. In both cases, MR increased the transepithelial electrical resistance across the epithelium, while decreasing passive leak of small nonelectrolytes. However, the specific target protein modifications involved were unique to each case. Overall, this provides an example of the primary level on which MR functions to modify, and improve, an organism.

Keywords: barrier function; claudin; methionine; occludin; tight junction; transepithelial.

Figure 1

Diagrammatic cross section of a simple epithelial barrier. Reproduced with permission from Ref. .

Figure 2

Intracellular sulfur-containing amino acid levels decline sharply in sulfur-containing, amino acid–restricted culture medium. LLC-PK₁ cell layers were placed under either control (100% cysteine/100% cystine/100% methionine) (red bar) or sulfur-containing amino acid–restricted (50% cysteine/0% cystine/10% methionine) (blue bar) conditions for 1 week before being harvested and amino acid pools analyzed. Reproduced with permission from Ref. .

Figure 3

Sulfur-containing, amino acid–restricted cell sheets exhibit a significant increase in transepithelial electrical resistance and a significant decrease in transepithelial ¹⁴C-mannitol flux. LLC-PK₁ cell layers were kept on control (100% cysteine/100% cystine/100% methionine) (red bar) or sulfur-containing, amino acid–restricted (50% cysteine/0% cystine/10% methionine) (blue bar) conditions for 1 week before measuring transepithelial electrical resistance and transepithelial mannitol leak. Data shown represent the mean ± standard error of n = 9 cell sheets. Significance was determined by Student’s t-test, assuming equal variances. Reproduced with permission from Ref. .

Figure 4

Western immunoblots of integral tight junction proteins in sulfur-containing, amino acid–restricted (left) versus control (right) conditions. LLC-PK₁ cell layers were maintained at confluence in either control (100% cysteine/100% cystine/100% methionine) or sulfur-containing amino acid-restricted conditions (50% cysteine/0% cystine/10% methionine) for one week, then harvested as described. Immunoblots shown and the percentage increase/decrease represents the mean ± standard error, and was calculated by densitometry of the appropriate bands of 4 separate polyacrylamide gel electrophoresis and western immunoblots of the membrane fraction of cells. Significance was determined using Student’s t-test, assuming equal variances. Reproduced with permission from Ref. .

Figure 5

MR diet affects TJ barrier function. ¹⁴C-mannitol flux (pmole/min/cm²) across distal colon tissues from rats on control and MR diets for 28 days (n = 5 tissues per group). Values shown represent mean ± standard error. P < 0.05, Student’s t-test, two-tailed. Reproduced with permission from Ref. .

Figure 6

MR of the diet of Sprague-Dawley rats alters mRNA expression and protein abundance of certain tight junction components. (A) RT-PCR of specific tight junction protein mRNA in the distal colon of animals on control and methionine-restricted diet (n = 3). (B). Western blot analysis showing the steady state level of tight junction proteins (top panel: occludin; middle panel: claudin-3) in the distal colon of animals on control and MR diet (numbers indicate animal pairs). Sample loading was normalized to β-tubulin levels (bottom panel). Reproduced with permission from Ref. .