Methionine restriction prevents onset of type 2 diabetes in NZO mice

Abstract

Dietary methionine restriction (MR) is well known to reduce body weight by increasing energy expenditure (EE) and insulin sensitivity. An elevated concentration of circulating fibroblast growth factor 21 (FGF21) has been implicated as a potential underlying mechanism. The aims of our study were to test whether dietary MR in the context of a high-fat regimen protects against type 2 diabetes in mice and to investigate whether vegan and vegetarian diets, which have naturally low methionine levels, modulate circulating FGF21 in humans. New Zealand obese (NZO) mice, a model for polygenic obesity and type 2 diabetes, were placed on isocaloric high-fat diets (protein, 16 kcal%; carbohydrate, 52 kcal%; fat, 32 kcal%) that provided methionine at control (Con; 0.86% methionine) or low levels (0.17%) for 9 wk. Markers of glucose homeostasis and insulin sensitivity were analyzed. Among humans, low methionine intake and circulating FGF21 levels were investigated by comparing a vegan and a vegetarian diet to an omnivore diet and evaluating the effect of a short-term vegetarian diet on FGF21 induction. In comparison with the Con group, MR led to elevated plasma FGF21 levels and prevented the onset of hyperglycemia in NZO mice. MR-fed mice exhibited increased insulin sensitivity, higher plasma adiponectin levels, increased EE, and up-regulated expression of thermogenic genes in subcutaneous white adipose tissue. Food intake and fat mass did not change. Plasma FGF21 levels were markedly higher in vegan humans compared with omnivores, and circulating FGF21 levels increased significantly in omnivores after 4 d on a vegetarian diet. These data suggest that MR induces FGF21 and protects NZO mice from high-fat diet-induced glucose intolerance and type 2 diabetes. The normoglycemic phenotype in vegans and vegetarians may be caused by induced FGF21. MR akin to vegan and vegetarian diets in humans may offer metabolic benefits via increased circulating levels of FGF21 and merits further investigation.-Castaño-Martinez, T., Schumacher, F., Schumacher, S., Kochlik, B., Weber, D., Grune, T., Biemann, R., McCann, A., Abraham, K., Weikert, C., Kleuser, B., Schürmann, A., Laeger, T. Methionine restriction prevents onset of type 2 diabetes in NZO mice.

Keywords: energy expenditure; hyperglycemia; obesity; vegan; vegetarian.

Figure 1

MR prevents hyperglycemia and improves glucose homeostasis in NZO mice. A) Study design. At the age of 3 wk, NZO mice were placed on a Con diet without cystine, or a Con (+Cys) diet for 1 wk, at which point a random subgroup of animals was transferred to an MR diet [MR or MR (+Cys), respectively] for 9 wk. B, C) Final random blood glucose (B) and plasma insulin concentrations (C). Six weeks after the dietary switch, an OGTT (glucose 2 mg/g body weight by oral gavage) was performed in mice unfed for 6 h. D, E) Blood glucose (D) and fasting blood glucose (E) during OGTTs. F–H) Plasma insulin (F), fasting plasma insulin (G), and area under the curve of insulin levels (H) during OGTTs. I) Insulin sensitivity index calculated by Matsuda. J) Circulating FGF21 levels. K) Final gene expression of Fgf21 in indicated tissues. L, M) Final circulating leptin (L) and total adiponectin levels (M) assessed by ELISA. N) Final total pancreatic insulin. Quad., quadriceps. Data are presented as means ± sem (n = 6–8/group). Differences vs. the Con group were calculated by 1-way ANOVA [with Bonferonni post hoc analyses (B, C, E, G–I, L, M)], 2-way ANOVA [with Bonferonni post hoc analyses (D, F, J)], and a 2-tailed Student’s t test (K, N). ^#P ≤ 0.1 and ^#P < 0.05; *P ≤ 0.05, **P ≤ 0.01.

Figure 2

Switching to a vegetarian diet increases FGF21 in humans. A) Correlations between plasma FGF21 and methionine levels of NZO mice. B–D) Plasma FGF21 concentrations of omnivore (n = 36) or vegan (n = 36) humans (B), of omnivore (n = 16) or vegetarian (n = 12) humans at baseline (C), and omnivore human subjects (n = 16) given a vegetarian diet for 4 d (D). Black circles, Con; white circles, MR. Data are presented as means ± sem. Pearson correlation (A); 2-tailed Student’s t test for difference between groups (B, C); and paired, 2-tailed Student’s t test (D). *P ≤ 0.05, **P ≤ 0.01.

Figure 3

MR lowers weight gain and delays growth in NZO mice. Mice were treated as described in Fig. 1. A) Weekly body weight. B, C) Percentage body fat (B) and percentage body lean (C) at the beginning and end of the study. D) Final body length. Data are presented as means ± sem (n = 6–8/group). Differences vs. the Con group were calculated by 2-way ANOVA with Bonferonni post hoc analyses (A–C) and 2-tailed Student’s t test (D). *P ≤ 0.05, **P ≤ 0.01.

Figure 4

MR increases EE in NZO mice. Mice were treated as described in Fig. 1. A) EE normalized to body weight in NZO mice consuming Con or MR diets for 8 wk. B–D) Mean EE normalized to body weight (B) or lean mass (C), and mean activity (D) for indicated periods throughout the 8 wk after diet switch. E–H) RER (E), mean RER for indicated periods (F), mean food intake (G), and water intake (H) for indicated periods throughout the eighth week after diet switch. Data are presented as means ± sem (n = 4/group). Differences vs. the Con group were calculated by a 2-tailed Student’s t test (B–D, F–H). *P ≤ 0.05, **P ≤ 0.01.

Figure 5

Dietary MR decreases hepatic fat storage and improves hepatic insulin sensitivity in NZO mice. Mice were treated as described in Fig. 1. Nine weeks after the diet switch, mice unfed for 6 h were treated intraperitoneally with NaCl or insulin (7 IU/kg body weight) 15 min before killing. A) Final weight of indicated organs. B, C) Final liver triacylglycerol (B) and glycogen content (C). D, E) Final plasma NEFA (D) and triacylglycerol (E). F) Hematoxylin and eosin staining of liver revealed morphologic changes consistent with triacylglycerol accumulation in Con mice. Scale bars, 100 µm. G, H) Western blots of total and phosphorylated Akt (G) and total PEPCK (H) in liver. Detecting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. gWAT, gonadal white adipose tissue; Panc., pancreas; Quad., quadriceps. Data are presented as means ± sem (n = 3–6/group). Differences between the groups were calculated by a 2-tailed Student’s t test. *P ≤ 0.05, **P ≤ 0.01.