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. 2016 Jul 4:6:28775.
doi: 10.1038/srep28775.

Metformin inhibits Branched Chain Amino Acid (BCAA) derived ketoacidosis and promotes metabolic homeostasis in MSUD

Davis S Sonnet  1 , Monique N O'Leary  1   2 , Mark A Gutierrez  3 , Steven M Nguyen  1 , Samiha Mateen  1 , Yuehmei Hsu  1 , Kylie P Mitchell  1 , Antonio J Lopez  4 , Jerry Vockley  5 , Brian K Kennedy  1 , Arvind Ramanathan  1
Affiliations

Metformin inhibits Branched Chain Amino Acid (BCAA) derived ketoacidosis and promotes metabolic homeostasis in MSUD

Davis S Sonnet et al. Sci Rep. .

Abstract

Maple Syrup Urine Disease (MSUD) is an inherited disorder caused by the dysfunction in the branched chain keto-acid dehydrogenase (BCKDH) enzyme. This leads to buildup of branched-chain keto-acids (BCKA) and branched-chain amino acids (BCAA) in body fluids (e.g. keto-isocaproic acid from the BCAA leucine), leading to numerous clinical features including a less understood skeletal muscle dysfunction in patients. KIC is an inhibitor of mitochondrial function at disease relevant concentrations. A murine model of intermediate MSUD (iMSUD) shows significant skeletal muscle dysfunction as by judged decreased muscle fiber diameter. MSUD is an orphan disease with a need for novel drug interventions. Here using a 96-well plate (liquid chromatography- mass spectrometry (LC-MS) based drug-screening platform we show that Metformin, a widely used anti-diabetic drug, reduces levels of KIC in patient-derived fibroblasts by 20-50%. This Metformin-mediated effect was conserved in vivo; Metformin-treatment significantly reduced levels of KIC in the muscle (by 69%) and serum (by 56%) isolated from iMSUD mice, and restored levels of mitochondrial metabolites (e.g. AMP and other TCA). The drug also decreased the expression of mitochondrial branched chain amino transferase (BCAT) which produces KIC in skeletal muscle. This suggests that Metformin can restore skeletal muscle homeostasis in MSUD by decreasing mitochondrial KIC production.

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Figures

Figure 1
Figure 1
Levels of KIC and mitochondrial markers in MSUD (a) BCAA catabolic pathway (b) Image of skeletal muscle from WT and iMSUD mice. (c) Analysis of mean fiber cross-sectional area in quadriceps and gastrocnemius muscles. P values were assessed by unpaired t-test with Welch’s correction (*p ≤ 0.05). Results are shown as mean ± SEM of independent animals (n = 3). (d) Concentration of KIC and leucine measured in iMSUD and WT mice serum. Results are shown as mean ± SEM of independent animals (n ≥ 4). Statistical significance was determined using unpaired two-tailed Students’ t-test and is denoted by *p ≤ 0.05. (e) Concentration of KIC and leucine measured in iMSUD and WT mice muscle. Results are shown as mean ± SEM of independent animals (n ≥ 3). Statistical significance was determined using unpaired two-tailed Students’ t-test and is denoted by ***p ≤ 0.001. (f) LC-MS based metabolic profiling of mitochondrial metabolites observed in WT and iMSUD mice. Heat map includes relative values baselined to wild-type of independent animals (n ≥ 3). (g) NRF2, PGC, GCLC, GCLC, GSR, TFAM, COXII, and ANT1 gene expression in WT and iMSUD mice. Indicated mRNA levels were determined by qPCR. Bar graphs indicate mean ± SEM of independent animals (n ≥ 4). *p ≤ 0.05 (h) LC-MS based metabolic profiling of mitochondrial metabolites measured in C2C12 skeletal muscle cells treated with KIC (0.1–0.5 mM) for 48 h. Heat map includes relative values baselined to untreated C2C12 skeletal muscle cells of 4 independent preparations.(i) Extracellular α-ketoglutarate secreted by the C2C12 muscle cells treated with KIC (0.1–0.5 mM) for 48 h. Bar graphs indicate mean ± RSD of 4 independent preparations. Statistical significance was determined using unpaired two-tailed Students’ t-test and is denoted by ***p ≤ 0.001 and *p ≤ 0.05. (j) JC-1 fluorescence ratio of C2C12 skeletal muscle cells treated with KIC (0.25–01.0 mM) for 48 h. Data indicate mean ± SD of 4 independent preparations.
Figure 2
Figure 2
LC-MS based chemical screening of MSUD patient derived fibroblast reveals that Metformin diminishes the accumulation of KIC and induces a glycolytic switch (a) 96-well plate LC-MS based assay for screening drugs that modulate leucine metabolism in patient cells. MSUD fibroblasts were cultured in a 96-well plate and treated with an in-house curated collection of modulators of pathways in central carbon metabolism-AMPK, mTOR, fatty acid oxidation and oxidative phosphorylation. Following 72 h treatment, spent media was collected and diluted 10-fold for LC-MS analysis to measure levels of leucine and KIC in MSUD fibroblasts. (b) Extracellular KIC measured in patient derived MSUD cells (gm297, gm296) treated with Metformin (0.25–1 mM) and AICAR (25–100 μM) for 72 h. Heat map includes relative values baselined to untreated gm296 and gm297 of 2 independent preparations. (c) Extracellular KIC and leucine measured in Type IA and II patient derived MSUD cells treated with Metformin (0.5 mM) for 72 h. Heat map includes relative values baselined to untreated of 3 independent preparations.
Figure 3
Figure 3
Metformin inhibits BCAA catabolism and promotes metabolic homeostasis in iMSUD mice (a) JC-1 fluorescence ratio of C2C12 skeletal muscle cells treated with KIC (0.25–01.0 mM) ±Metformin (0.25 mM) for 48 h. Data indicate mean ± SD of 4 independent preparations. *p ≤ 0.05 (b) BCAT1 and BCAT2 gene expression of C2C12 muscle cells treated with Metformin (2.0 mM) for 48–72 h. Indicated mRNA levels were determined by qPCR. Data indicate mean ± SEM. *p ≤ 0.05 (c) BCAT2 gene expression WT and iMSUD mice fed a standard 5LG6 diet (Lab Diet) or 5LG6 with 0.1% (w/w) Metformin ad libitum. Indicated mRNA levels were determined by qPCR. Bar graphs indicate mean ± SEM of independent animals (n ≥ 4). Statistical significance was determined using unpaired two-tailed Students’ t-test and is denoted by *p ≤ 0.05. LC-MS profiling of BCAA metabolites measured in (d) skeletal muscle and (e) serum from WT and iMSUD fed a standard 5LG6 diet (Lab Diet) or 5LG6 with 0.1% (w/w) Metformin ad libitum. Results are shown as mean ± SEM of independent animals (n ≥ 3). Statistical significance was determined using unpaired two-tailed Students’ t-test and is denoted by *p ≤ 0.05. (f) LC-MS profiling of mitochondrial metabolites measured in skeletal muscle from WT and iMSUD mice fed a standard 5LG6 diet (Lab Diet) or 5LG6 with 0.1% (w/w) Metformin ad libitum. Heat map includes relative values baselined to WT of independent animals (n ≥ 3). (g) α-ketoglutarate measured in skeletal muscle from WT and iMSUD mice fed a standard 5LG6 diet (Lab Diet) or 5LG6 with 0.1% (w/w) Metformin ad libitum. Results are shown as mean ± SEM of independent animals (n ≥ 3). Statistical significance was determined using unpaired two-tailed Students’ t-test and is denoted by *p ≤ 0.05. (h) Mechanism of Metformin-induced inhibition of the BCAA pathway via mitochondrial BCAT2.
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