Decrease of disease-related metabolites upon fasting in a hemizygous knock-in mouse model (Mut-ko/ki) of methylmalonic aciduria

Abstract

Methylmalonyl-CoA mutase (MMUT) is part of the propionyl-CoA catabolic pathway, responsible for the breakdown of branched-chain amino acids, odd-chain fatty acids and the side-chain of cholesterol. Patients with deficient activity of MMUT suffer from isolated methylmalonic aciduria (MMAuria), frequently presenting in the newborn period with failure to thrive and metabolic crisis. Even well managed patients remain at risk for metabolic crises, of which one known trigger is acute illness, which may lead to poor feeding and vomiting, putting the patient in a catabolic state. This situation is believed to result in increased breakdown of propionyl-CoA catabolic pathway precursors, producing massively elevated levels of disease related metabolites, including methylmalonic acid and propionylcarnitine. Here, we used fasting of a hemizygous mouse model (Mut-ko/ki) of MMUT deficiency to study the role of induced catabolism on metabolite production. Although mice lost weight and displayed markers consistent with a catabolic state, contrary to expectation, we found strongly reduced levels of methylmalonic acid and propionylcarnitine in fasted conditions. Switching Mut-ko/ki mice from a high-protein diet to fasted conditions, or from a standard diet to a no-protein diet, resulted in similar reductions of methylmalonic acid and propionylcarnitine levels. These results suggest, in our mouse model at least, induction of a catabolic state on its own may not be sufficient to trigger elevated metabolite levels.

Keywords: Methylmalonic aciduria; catabolism; disease amelioration; fasting; methylmalonyl‐CoA mutase; mouse model.

FIGURE 1

Mut‐ko/ki mice display a reduction of metabolite levels upon 24‐hour fasting. A, Changes in body weight, B, MMA, C, C3/C2 and, D, alanine in Mut‐ki/wt (n = 4) and Mut‐ko/ki (n = 4) female mice before and following 24 hours of fasting. MMA, C3/C2 and alanine were measured in dried blood spots. All changes between basal and fasting conditions for Mut‐ko/ki are significant by at least P < .05, except MMA in dried blood spots (P = .14). Changes between basal and fasting conditions for Mut‐ki/wt are significant for body weight and alanine. Paired t‐tests were used for these analyses. At the time of experiment mice were 3.5 months of age. Baseline levels were determined 1 day before the food withdrawal

FIGURE 2

Mut‐ko/ki mice display a reduction of metabolite levels upon long‐term fasting. A, Changes in body weight, B, 3‐hydroxybutyrate (3‐HB) in whole blood, and C, alanine, D, MMA, and E, C3/C2 in dried blot spots were measured before, during (after 24 hours) and at the end of 48 hours of fasting in Mut‐ki/wt (n = 5) and Mut‐ko/ki (n = 6) females. In Mut‐ko/ki mice, MMA, C3/C2 and alanine levels decrease after 24 hours of fasting (P < .001) and stabilize, body weight decrease after 24 hours of fasting (P < .05), while 3‐HB increase at 24 hours (P < .001) and further increase at 48 hours (P < .001). Changes between basal and 24‐hour fasting conditions for Mut‐ki/wt are significant for body weight, 3‐hydroxybutyrate and alanine. Two‐way analysis of variance with Tukey's multiple comparison tests were used for these analyses. At the time of experiment mice were 4 months of age. Baseline levels were determined 17 days before the food withdrawal

FIGURE 3

Reduction of metabolite levels upon 48‐hour fasting in mice fed a 51% protein diet. A, Changes in body weight, B, 3‐hydroxybutyrate (3‐HB), C, MMA and D, C3/C2 in dried blood spots were measured before and at the end of 48 hours of fasting in Mut‐ki/wt (n = 5) and Mut‐ko/ki (n = 5) females. E, MMA in plasma was measured in ad libitum fed or after 48 hours of fasting in cohorts of four to five mice. For Mut‐ko/ki mice: basal levels were 671 to 1030 μM, and reduced to 32 to 47 μM following fasting. F, For measurement in urine: samples from the same genotypes were pooled when insufficient volume. Fasted mice had MMA of 851 to 1260 mmol/mol creatinine, compared to 4540 to 97 520 mmol/mol creatinine when not fasted. ¹¹ In both Mut‐ko/ki and Mut‐ki/wt mice, body weight decrease (P < .001) and 3‐HB levels increase (P < .01) under fasting. In Mut‐ko/ki mice, MMA and C3/C2 levels in blood decrease (P < .01 and P < .001, respectively). Paired t‐tests were used for these analyses. MMA in plasma decrease in Mut‐ko/ki mice (P < .001, unpaired t‐test). Mice were 6 months of age. Baseline levels were determined 7 to 8 days before the food withdrawal

FIGURE 4

Mut‐ko/ki mice show reduced metabolite levels after switching to a 0% protein diet. A, Changes in body weight as well as, B, MMA and C, C3/C2 in dried blood spots were measured before and after switching from regular chow to a 0% protein diet in Mut‐ki/wt (n = 3) and Mut‐ko/ki (n = 3) females. Diet change was initiated at day 40 of life, indicated as Time 0. The first time point corresponds to the baseline level under a regular chow. Weight loss is not significant (P < .05); MMA and C3/C2 changes are significant by at least P < .001 between day 40 and all other days for Mut‐ko/ki. Two‐way analysis of variance with Tukey's multiple comparison tests were used for these analyses. Mice were switched to a 0% protein diet at day 40