Tetradecylthiopropionic acid induces hepatic mitochondrial dysfunction and steatosis, accompanied by increased plasma homocysteine in mice

Abstract

Background: Hepatic mitochondrial dysfunction plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Methyl donor supplementation has been shown to alleviate NAFLD, connecting the condition to the one-carbon metabolism. Thus, the objective was to investigate regulation of homocysteine (Hcy) and metabolites along the choline oxidation pathway during induction of hepatic steatosis by the fatty acid analogue tetradecylthiopropionic acid (TTP), an inhibitor of mitochondrial fatty acid oxidation.

Methods: Mice were fed a control diet, or diets containing 0.3 %, 0.6 %, or 0.9 % (w/w) TTP for 14 days. Blood and liver samples were collected, enzyme activities and gene expression were analyzed in liver, lipid and fatty acid composition in liver and plasma, one-carbon metabolites, B-vitamin status, carnitine and acylcarnitines were analyzed in plasma.

Results: Liver mitochondrial fatty acid oxidation decreased by 40 % and steatosis was induced in a dose dependent manner; total lipids increased 1.6-fold in animals treated with 0.3 % TTP, 2-fold with 0.6 % TTP and 2.1 fold with 0.9 % TTP compared to control. The higher hepatic concentration of fatty acids was associated with shortening of carbon-length. Furthermore, the inhibited fatty acid oxidation led to a 30-fold decrease in plasma carnitine and 9.3-fold decrease in acetylcarnitine at the highest dose of TTP, whereas an accumulation of palmitoylcarnitine resulted. Compared to the control diet, TTP administration was associated with elevated plasma total Hcy (control: 7.2 ± 0.3 umol/L, 0.9 % TTP: 30.5 ± 5.9 umol/L) and 1.4-1.6 fold increase in the one-carbon metabolites betaine, dimethylglycine, sarcosine and glycine, accompanied by changes in gene expression of the different B-vitamin dependent pathways of Hcy and choline metabolism. A positive correlation between total Hcy and hepatic triacylglycerol resulted.

Conclusions: The TTP-induced inhibition of mitochondrial fatty acid oxidation was not associated with increased hepatic oxidative stress or inflammation. Our data suggest a link between mitochondrial dysfunction and the methylation processes within the one-carbon metabolism in mice.

Fig. 1

Metabolites of the homocysteine remethylation and sulfoxation, transsulfuration, and choline oxidation pathways. Names of enzymes are beside the arrows. Vitamins involved in the enzymatic reactions are shown in green circles. Abbreviations: BADH, betaine aldehyde dehydrogenase; BHMT, betaine-homocysteine methyltransferase; CBS, cystathionine beta-synthase; CHDH, choline dehydrogenase; CTH, cystathionine gamma-lyase; DMG, dimethylglycine; DMGDH, dimethylglycine dehydrogenase; GNMT, glycine N-methyltransferase; Hcy, homocysteine; MAT, methionine adenosyltransferase; Met, methionine; MetSO, methionine sulfoxide; 5-mTHF, 5-methyltetrahydrofolate; MTs, methyltransferases; MTR, methionine synthase; MSR (encoded by the gene Mtrr), methionine synthase reductase; PC, phosphatidylcholine; PE, phosphatidyletanolamine; PEMT, phosphatidyl-ethanolamine methyltransferase; SAH, s-adenosylhomocysteine; SAM, s-adenosylmethionine; SARDH, sarcosine dehydrogenase; SHMT1, serine hydroxymethyltransferase 1; SHMT2, serine hydroxymethyltransferase 2; THF, tetrahydrofolate; TML, trimethyllysine

Fig. 2

Lipid droplet morphology in frozen liver tissue sections from tetradecylthiopropionic acid (TTP) treated mice. Representative images of Oil-red-O stained liver sections from male C57BL/6 mice fed a control low-fat diet, or low-fat diets supplemented with 0.3 % (w/w), 0.6 % or 0.9 % TTP for 2 weeks (n = 3)

Fig. 3

Hepatic lipid profile with increasing dose of tetradecylthiopropionic acid (TTP). Male C57BL/6 mice were fed a low-fat control diet (black), or low-fat diets supplemented with 0.3 % (grey) (w/w), 0.6 % (dark grey) or 0.9 % (light grey) TTP for 2 weeks. Levels of (a) total hepatic lipids (combination of triacylglycerol (TAG), cholesterol, and phospholipids), (b) TAG, (c) cholesterol, and (d) phospholipids were as indicated. Mean values with SD are presented (n = 7-8), and statistically significant difference from control was determined by one-way ANOVA with Dunnett’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 4

The effect of tetradecylthiopropionic acid (TTP) on hepatic lipid metabolism in mice. Male C57BL/6 mice were fed a low-fat control diet (black), or low-fat diets supplemented with 0.3 % (grey) (w/w), 0.6 % (dark grey) or 0.9 % (light grey) TTP for 2 weeks. a β-oxidation as measured by degradation of palmitoyl-CoA in fresh liver homogenates. b Inhibition of the β-oxidation reaction in the presence of malonyl-CoA. The activities of (c) Carnitine palmitoyl transferase (CPT)-I, (d) CPT-II, (e) HMG-CoA synthase, (f) citrate synthetase, (g) fatty acyl-CoA oxidase (ACOX1), and (h) acyl-CoA synthase were measured in frozen liver homogenates. Mean values with SD are presented (a–c: n = 5 – 7, d–i: n = 7–8), and statistically significant difference from control was determined by one-way ANOVA with Dunnett’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 5

The effect of tetradecylthiopropionic acid (TTP) on plasma L-carnitine precursors, free L-carnitine, and L-carnitine esters. Male C57BL/6 mice were fed a low-fat control diet (black), or low-fat diets supplemented with 0.3 % (grey) (w/w), 0.6 % (dark grey) or 0.9 % (light grey) TTP for 2 weeks, and (a) L-carnitine, (b) γ-butyrobetaine, (c) palmitoylcarnitine, (d) octanoylcarnitine, (e) propionylcarnitine, (f) (iso)valerylcarnitine, and (g) acetylcarnitine was measured in fasting plasma samples. Mean values with SD are presented (pooled samples from 2-3 mice, n = 3), and statistically significant difference from control was determined by one-way ANOVA with Dunnett’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 6

The effect of tetradecylthiopropionic acid (TTP) on antioxidative and anti-inflammatory parameters. Male C57BL/6 mice were fed a low-fat control diet (black), or low-fat diets supplemented with 0.3 % (grey) (w/w), 0.6 % (dark grey) or 0.9 % (light grey) TTP for 2 weeks. Levels of (a) plasma total antioxidant capacity, (b) plasma anti-inflammatory fatty acid index, (c) liver anti-inflammatory fatty acid index, (d) plasma double bond index (DBI), (e) liver DBI were as indicated. Mean values with SD are presented (a: n = 3–8; b–e: n = 7–8), and statistically significant difference from control was determined by one-way ANOVA with Dunnett’s post hoc test (*P < 0.05)

Fig. 7

The effect of tetradecylthiopropionic acid (TTP) on plasma one-carbon metabolites in mice. Male C57BL/6 mice were fed a low-fat control diet (black), or low-fat diets supplemented with 0.3 % (grey) (w/w), 0.6 % (dark grey) or 0.9 % (light grey) TTP for 2 weeks, and (a) sarcosine, (b) betaine, (c) dimethylglycine (DMG), (d) glycine, (e) serine, (f) methionine (Met), (g) Met-sulfoxide, (h) total homocysteine (tHcy), (i) cystathionine, (j) total cysteine, and (k) trimethyllysine was measured in fasting plasma samples. Mean values with SD are presented (pooled samples from 2–3 mice, n = 3), and statistically significant difference from control was determined by one-way ANOVA with Dunnett’s post hoc test (*P < 0.05, **P < 0.01). l There was a positive association between hepatic TAG and plasma tHcy as demonstrated by linear regression

Fig. 8

The effect of tetradecylthiopropionic acid (TTP) on plasma B-vitamins and derivatives. Male C57BL/6 mice were fed a low-fat control diet (black), or low-fat diets supplemented with 0.3 % (grey) (w/w), 0.6 % (dark grey) or 0.9 % (light grey) TTP for 2 weeks, and (a) flavin mononucleotide, (b) riboflavin, (c) nicotinamide, (d) pyridoxal 5′-phosphate, (e) pyridoxal, (f) 4-pyridoxic acid, and (g) methylmalonic acid (MMA), was measured in fasting plasma samples. Mean values with SD are presented (pooled samples from 2-3 mice, n = 3), and statistically significant difference from control was determined by one-way ANOVA with Dunnett’s post hoc test (*P < 0.05)