Cysteine restriction-specific effects of sulfur amino acid restriction on lipid metabolism

Abstract

Decreasing the dietary intake of methionine exerts robust anti-adiposity effects in rodents but modest effects in humans. Since cysteine can be synthesized from methionine, animal diets are formulated by decreasing methionine and eliminating cysteine. Such diets exert both methionine restriction (MR) and cysteine restriction (CR), that is, sulfur amino acid restriction (SAAR). Contrarily, SAAR diets formulated for human consumption included cysteine, and thus might have exerted only MR. Epidemiological studies positively correlate body adiposity with plasma cysteine but not methionine, suggesting that CR, but not MR, is responsible for the anti-adiposity effects of SAAR. Whether this is true, and, if so, the underlying mechanisms are unknown. Using methionine- and cysteine-titrated diets, we demonstrate that the anti-adiposity effects of SAAR are due to CR. Data indicate that CR increases serinogenesis (serine biosynthesis from non-glucose substrates) by diverting substrates from glyceroneogenesis, which is essential for fatty acid reesterification and triglyceride synthesis. Molecular data suggest that CR depletes hepatic glutathione and induces Nrf2 and its downstream targets Phgdh (the serine biosynthetic enzyme) and Pepck-M. In mice, the magnitude of SAAR-induced changes in molecular markers depended on dietary fat concentration (60% fat >10% fat), sex (males > females), and age-at-onset (young > adult). Our findings are translationally relevant as we found negative and positive correlations of plasma serine and cysteine, respectively, with triglycerides and metabolic syndrome criteria in a cross-sectional epidemiological study. Controlled feeding of low-SAA, high-polyunsaturated fatty acid diets increased plasma serine in humans. Serinogenesis might be a target for treating hypertriglyceridemia.

Keywords: aging; caloric restriction; cysteine; metabolic syndrome; methionine; nutrition; sulfur amino acids; triglycerides.

FIGURE 1

Methionine restriction and cysteine restriction exert discrete effects on morphometry and plasma hormone concentrations. Eight‐week‐old male F344 rats were fed CD, SAAR, MR (MR1, MR2, MR3, MR4), and CR (CR1, CR2, CR3, CR4, CR5) diets for 12 weeks. Changes in growth rate (a), food intake (b), plasma Igf1 (c), plasma Fgf21 (d), and plasma leptin (e) were dependent on MR, while changes in plasma adiponectin (f) were dependent on CR. Two‐tailed Student's t‐test was used to find differences (represented by μ) between CD and SAAR groups (n = 16). Dose responses to MR‐ and CR‐titrated diets were analyzed by simple linear regression (coefficients represented by MR_β and CR_β; n = 8/group); error bars represent the means and standard error of means. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. = not significant. White and black triangles below the x‐axes represent the SAA gradient in MR and CR diets, respectively

FIGURE 2

Methionine restriction and cysteine restriction induce distinct changes in plasma amino acid concentrations. Although SAAR changed plasma concentrations of glutamic acid (a), glycine (b), and lysine (c), these amino acids did not show dose response to either MR or CR. A strong dose response was exhibited by plasma histidine (d), methionine (e), phenylalanine (f), serine (g), threonine (h), and tryptophan (i) to CR. For sample sizes, statistics, and annotations refer to Figure 1

FIGURE 3

Cysteine restriction, but not methionine restriction, induces hepatic de novo serine biosynthesis. CR, but not MR, increased hepatic serine concentrations (a), Phgdh mRNA (b, c), and Phgdh protein levels (d, e). For statistics and annotations refer to Figure 1. In panel a, n = 16 for CD and SAAR groups, n = 8/group for all other groups; in all other panels, n = 4–8/group; in panel d, only the diets with the highest and lowest concentration of Met (MR1 and MR4) and Cys (CR1 and CR5) were probed. μ^c—cannot determine (due to the absence of bands, numbers for band densities were not available)

FIGURE 4

Cysteine restriction increases serinogenesis at the expense of glyceroneogenesis. MR, but not CR, decreased blood glucose (a). Neither MR nor CR altered the mRNA expression of G6pc (b, c) and Pck1 (d, e). CR, but not MR, increased the hepatic mRNA expression of Pck2 (f, g) and its protein Pepck‐M (h, i), and decreased hepatic glycerol‐3‐phosphate concentration (j). For statistics and annotations refer to Figure 1. In panel a, n = 14 for CD and SAAR groups, n = 8/group for all other groups; in all other panels, n = 4–8/group

FIGURE 5

SAAR‐induced changes in molecular markers of serinogenesis and lipid metabolism are dependent on sex, dietary fat content, and age‐at‐onset. Young (8‐week‐old, circles) and adult (18‐month‐old, triangles) male (blue circles) and female (red circles) C57BL/6J mice were fed CD and SAAR diets with either 10% fat or 60% fat for at least 3 months. Statistical significance of SAAR‐induced changes in hepatic glutathione (a–c), hepatic protein expressions of Nrf2 (d–f), Phgdh (g–i), and Pck2 (j–l), plasma triglycerides (m–o), and perigonadal adipose tissue weights (p–r) were analyzed by 2‐way ANOVA. The interaction of SAAR‐induced changes with dietary fat in young mice and sex in adult mice are indicated by μ_int. Pairwise comparisons between CD and SAAR are indicated by μ. n = 8/group; PGAT—perigonadal adipose tissue; error bars indicate means and standard error of means. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. = not significant

FIGURE 6

Associations between cysteine, serine, and triglycerides in epidemiological and short‐term human feeding studies reflect mechanistic findings from rodent studies. 1) Plasma tCys, Ser, and tCys/Ser correlate with plasma triglycerides and the number of MetS criteria in humans. Log‐transformed plasma concentrations of Met (a, e), tCys (b, f), Ser (c, g), and tCys/Ser (d, h) were plotted against log‐transformed triglycerides (a–d) and MetS criteria (e–h), respectively. Red and blue lines in a‐d represent unadjusted and adjusted (for age, sex, and BMI) regression lines, respectively. n = 307 for plasma Met and tCys and n = 287 for plasma Ser; box plots represent the distribution of unadjusted amino acid concentrations within each category of MetS. Numbers in parenthesis after β_un and β_ad represent regression coefficients from unadjusted and adjusted models, respectively. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. = not significant). (2) Plasma Ser levels were amenable to low SAA diets depending on dietary fatty acid composition. (i) Change in plasma serine in overweight/obese women (n = 13) on a 7‐day pilot dietary intervention trial with low (SAA_low) or high (SAA_high) sulfur amino acid content. p‐Values represent group x time interactions (change in plasma serine over time between groups) and were computed with linear mixed models adjusted for baseline levels of serine. (b) Change in plasma serine in normal‐weight individuals (n = 14) participating in a 7‐day pilot dietary intervention trial with high polyunsaturated fatty acid contents and low SAA concentrations (SAA_low+PUFA) versus a diet high in saturated fatty acids and high sulfur amino acids (SAA_high+SFA). Red and blue dots represent mean Ser concentrations on high‐ and low‐SAA diets, respectively. Error bars represent confidence intervals