Fig 1.
Simplified TCA cycle and anaplerosis in CNS and muscle.
Red numbers indicate anaplerotic pathways, that can refill the levels of C4 intermediates of the cycle: 1 pyruvate carboxylase (mostly in CNS), 2 propionyl-CoA carboxylase, and 3 glutamic pyruvic transaminases (very important in muscle). C5 ketones, branched chain amino acids and heptanoate, are metabolized to propionyl-CoA and can therefore be anaplerotic via the propionyl-CoA carboxylation pathway. OAA–oxaloacetate, 2-OG– 2-oxoglutarate, Ile–isoleucine, Val—valine.
Table 1.
Gene names, symbol, forward and reverse primer sequences and of primers used for the gene expression studies of metabolic genes.
Fig 2.
Triheptanoin preserves motor neurons.
Starting at P35, female wild-type and hSOD1G93A mice were either treated with triheptanoin (TRIH) or control (CON) diet until P70. (A) Stereologically counted motor neuron numbers in the L4-L5 segments of these 70 day old mice (n = 7–10) revealed a 38% loss of motor neurons in control-fed hSOD1G93A mice. Triheptanoin provided a 33% protection against motor neuron loss. Two way ANOVA p<0.0001 for genotype, p = 0.0126 for treatment, the stars indicate results from a Bonferroni multiple comparisons post hoc tests if significant (**** p<0.0001, * p<0.05) (B-D) Representative thionine stained spinal cord sections from an untreated wild-type mouse (B) and untreated (C) and triheptanoin-treated (D) hSOD1G93A mice with arrows pointing to the motor neurons counted. Scale bar 100 μm.
Fig 3.
Triheptanoin treatment delays the loss of hind limb grip strength in hSOD1G93A mice.
Starting at P35, female wild-type and hSOD1G93A mice were treated with triheptanoin (TRIH) or control (CON) diet until they were sacrificed. (A) No differences in grip strength was observed between triheptanoin (green open triangles, n = 15) and control treated wild-type mice (black filled squares, n = 12). (B) The grip strength over time differed in triheptanoin treated (red crosses, n = 8) vs. untreated (blue empty circles, n = 5) hSOD1G93A mice (p = 0.04, two way ANOVA), with treated mice having higher grip strength at 18 and 19.5 weeks (p<0.05 Bonferroni post-hoc test). (C) The onset of hind limb grip strength loss was delayed by 2.8 weeks in triheptanoin treated hSOD1G93A mice when compared to untreated hSOD1G93A mice (p = 0.002, t-test). (D) Overall hind limb grip strength shown as the area under the curve over time was increased in triheptanoin treated hSOD1G93A mice compared to control treated hSOD1G93A mice (p = 0.02, t-test). (E) The onset of balance loss in triheptanoin treated hSOD1G93A mice was significantly delayed by 13 days (p = 0.0016, t-test). (F) Body weights over time were significantly different between triheptanoin treated vs. untreated wild-type mice and treated vs. untreated hSOD1G93A mice. (G) The onset of body weight loss in triheptanoin treated vs. untreated hSOD1G93A mice (n = 5) was delayed (p = 0.007, t-test). * p<0.05, ** p<0.01. The onset of body weight loss was defined as the day where a loss of more than 10% in an individual mouse occurred relative to the mean body weight from week 12 to 17 was observed. Also all subsequent three body weight measurements were ≤ 90% of the original mean weight.
Fig 4.
Gapdh, Pdha, Ogdh and Sdha mRNA expression in gastrocnemius muscle after triheptanoin treatment.
Starting at P35, female wild-type and hSOD1G93A mice were treated with triheptanoin (TRIH) or control (CON) diet until they were sacrificed. Quantitative real time PCR analysis of Gapdh, Pdha1, Ogdh and Sdha of the gastrocnemius muscle of 10 and 25 week old wild-type and hSOD1G93A mice untreated or treated with triheptanoin relative to house keeping genes. N-numbers of each group used in all graphs are indicated in the top bar graphs. The insets above each graph show the p-values for the effects of genotype in two-way ANOVAs, while the effect of diet was p>0.05 for each bar graph. When significant, the results of Bonferroni post tests are shown by stars (* p<0.05, ** p<0.01), showing that decreases of mRNA levels of several enzymes in hSOD1G93A mice were not apparent with triheptanoin treatment.
Fig 5.
Relative expression of the anaplerotic genes, pyruvate carboxylase Pcx and glutamic pyruvic transferases Gpt1 and 2.
Starting at P35, female wild-type and hSOD1G93A mice were either treated with triheptanoin (TRIH) or control (CON) diet until they were sacrificed. Gene expresssion is compared in the gastrocnemius muscle of 10 and 25 week old triheptanoin treated vs. untreated wild-type and hSOD1G93A mice relative to housekeeping genes. N-numbers of each group used in all graphs are indicated in the top bar graphs. The insets above each graph show the p-values for the effects of genotype in two-way ANOVAs, while the effect of diet was p>0.05 for each bar graph. When significant, the results of Bonferroni post tests are shown by a star (* p<0.05), indicating that triheptanoin treatment prevented the decrease of expression in Gpt2 mRNA.
Fig 6.
Gene expression of enzymes involved in the propionyl-CoA carboxylase pathway.
Starting at P35, female wild-type and hSOD1G93A mice were treated with triheptanoin (TRIH) or control (CON) diet until they were sacrificed. Relative expression of the α (Pcca) and β (Pccb) subunits of propionyl-CoA carboxylase and methylmalonyl mutase (Mut). Expresssion is compared in the gastrocnemius muscle of 10 and 25 week old wild-type and hSOD1G93A mice untreated or treated with triheptanoin relative to housekeeping genes. N-numbers used for each group throughout the experiments are indicated in the top bar graphs. The insets above each graph show the p-values for the effects of genotype in two-way ANOVAs, while the effect of diet was p>0.05 for each bar graph. When significant, the results of Bonferroni post tests are indicated by a star (* p<0.05), indicating that triheptanoin treatment in hSOD1G93A mice protected against lowered expression of Pccb and Mut mRNA.
Fig 7.
Lower maximal activities of 2-oxoglutarate dehydrogenase (OGDH) in hSOD1G93A gastrocnemius muscle.
The maximal activities of OGDH in extracts from gastrocnemius muscle of male wild-type and hSOD1G93A mice at different disease stages are compared. Stages are defined as presymptomatic (days 35–36), onset (days 63–75), mid-stage (days 110–130) and end-stage (days 150–175). The inset above the graph shows the two-way ANOVA p-values for the effects of genotype and disease stage, indicating that 2-oxoglutarate activity in hSOD1G93A mice declines with progression of disease. The star denotes significance in the Bonferroni post test (* p<0.05).
Fig 8.
Triheptanoin treatment increased levels of plasma β-hydroxybutyrate.
Plasma β-hydroxybutyrate levels (mM) in 70 days old wild-type and hSOD1G93A mice fed with either triheptanoin (TRIH) or control (CON) diet from day 35 to 70 (One-way ANOVA p = 0.042; Fisher’s LSD post test: *p<0.05, ***p<0.001, n = 10–12).
Fig 9.
Hypothesized mechanisms of triheptanoin.
Triheptanoin is the triglyceride of heptanoate, which is metabolized to acetyl-CoA as well as propionyl-CoA providing alternative and anaplerotic fuel. Following carboxylation propionyl-CoA produces succinyl-CoA (anaplerosis), which via metabolism to oxaloacetate can increase ATP production and aid in further acetyl-CoA oxidation. Thus triheptanoin can improve mitochondrial energy production and thereby protect neurons and muscle against degeneration.