Dodecanedioic acid prevents and reverses metabolic-associated liver disease and obesity and ameliorates liver fibrosis in a rodent model of diet-induced obesity

Abstract

Dodecanedioic acid (DC12) is a dicarboxylic acid present in protective polymers of fruit and leaves. We explored the effects of DC12 on metabolic dysfunction-associated steatohepatitis (MASH) and obesity. DC12 supplementation (100 mg/kg/day) was added to a high-fat diet (HFD) for 8 weeks in rodents to assess its impact on obesity and MASH prevention. Rats given DC12 experienced significant reductions of weight gain, liver and visceral fat weight, and improved glucose tolerance and insulin sensitivity. Liver histology showed protection against diet-induced MASH, with reduced steatosis, hepatocyte ballooning, and fibrosis. For weight-loss and MASH reversion, rats were fed HFD for 14 weeks, followed by 6 weeks with or without DC12. DC12 supplementation (100 mg/kg/day) led to a significant reduction of weight gain and liver weight. DC12 induced white adipose tissue beiging and reduced adiposity with a decrease of visceral fat. It also improved glucose tolerance, insulin sensitivity, and reduced hepatic gluconeogenic gene expression. Liver histology revealed a significant reduction in steatosis, hepatocyte ballooning, and inflammation as well as fibrosis, indicating MASH reversal. DC12 reduced hepatic lipogenesis enzymes as well as de novo lipogenesis measured by deuterated water and increased fatty acid β-oxidation. Plasma lipid profile showed lower triglycerides and phosphatidylcholines in the DC12 group. Notably, DC12 decreased mINDY expression, the cell membrane Na+-coupled citrate transporter, reducing citrate uptake and de-novo lipogenesis, linking its effects to improved lipid metabolism and reduced steatosis. We found that during the hepatic first pass, half of the DC12 ingested with water was taken up by the liver. The concentration of DC12 in the portal vein falls within the range identified in vitro as sufficient to inhibit citrate transport in hepatocytes.

Keywords: citrate; de novo lipogenesis; dodecanedioic acid (DC12); glucose tolerance; metabolic dysfunction‐associated steatohepatitis (MASH); obesity.

FIGURE 1

DC12 prevents obesity and MASH in DIO rodents. (A) Design of the prevention study. (B) Body weight time course. (C, D) Liver and visceral adipose tissue (VAT) weight. (E, F) Time courses of blood glucose and plasma insulin concentrations during an oral glucose tolerance test. (G) HOMA‐IR index. (H–O) Representative Hematoxylin and Eosin (H&E). (H, I) Oil Red O (ORO). (J, K) Picro Sirius Red. (L, M) Masson's Trichrome. (N, O) staining of liver sections from rats fed HFD with or without DC12 supplementation. Data are reported as mean value ± SEM of n = 10 animals per group. Statistical significances were calculated by unpaired two‐tailed t‐test and one‐way Anova with Bonferroni's correction for multiple comparisons, where appropriate. ****< 0.0001; ***< 0.0003; **< 0.003; *< 0.02.

FIGURE 2

DC12 promotes weight loss and insulin sensitivity. (A) Design of the MASH reversion study. (B) Body weight time course. (C) Visceral adipose tissue (VAT) weight. (D, E) Representative Hematoxylin and Eosin (H&E) staining of subcutaneous adipose tissue (SAT). (F) Gene expression of SAT beiging‐associated gene expression, namely PPARG coactivator 1 alpha (Pgc1a), Uncoupling protein 1 (Ucp1), and PR/SET domain 16 (Prdm16). (G, H) Time courses of blood glucose and plasma insulin concentrations during an oral glucose tolerance test. (I) HOMA‐IR index. (J) Liver weight. Data are reported as mean ± SEM of n = 10 animals per group. Statistical significances were calculated by unpaired two‐tailed t‐test and one‐way Anova with Bonferroni's correction for multiple comparisons, where appropriate. ****< 0.0001; ***< 0.0002; **< 0.004; *< 0.04.

FIGURE 3

DC12 reverse MASH. (A–H) Representative Hematoxylin and Eosin (H&E) (A, B), Oil Red O (ORO) (C, D), and Picro Sirius Red (E, F), Masson's Trichrome (G, H) staining of liver sections from rats fed HFD with or without DC12 supplementation. (I) Gene expression of hepatic fibrosis marker, namely alpha smooth muscle Actin (ASMA), Collagen Type I Alpha 1 Chain (COLA1A), and transforming growth factor‐beta (TGFβ). (J) Gene expression of hepatic key rate‐limiting enzymes of de novo lipogenesis, namely ATP‐citrate lyase (ACLY), acetyl‐CoA carboxylase (ACC), fatty acid synthase (FAS), and Diacylgycerol acyltransferase (DGAT). (K, L) Gene and protein expression of Carnitine Palmitoyltransferase 1A (CPT1A), the rate‐limiting enzyme of fatty acid β‐oxidation. Data are reported as mean ± SEM of n = 10 animals per group. Statistical significances were calculated by unpaired two‐tailed t‐test. ****< 0.0001; ***< 0.0002; **< 0.002.

FIGURE 4

Plasma lipidomic. Volcano plot showing the most significant lipid species found by univariate analysis. Gray values indicate those lipid species that are not significantly changed (p > .05). Negative values (in violet) indicate downregulated lipid species, while positive (in orange) values reflect upregulated lipids in rats fed HFD and DC12 (p < .05).

FIGURE 5

DC12 reduces citrate uptake through mINDY inhibition. (A, B) Hepatic gene and protein expression of mINDY in rats fed HFD with or without DC12 supplementation. (C) Plasma citrate concentration in rats fed HFD with or without DC12 supplementation. (D, E) Gene and protein expression of mINDY in human primary hepatocytes treated with palmitic acid (400 μM) in the presence or absence of DC12 (100 μM). (F) In vitro citrate uptake using increasing concentrations of DC12 (50–1000 μM); citrate uptake fit in the presence of increasing concentrations of DC12 with a 90% confidence band (shaded area). The following are the estimates of the parameters of the model: K _min = 19.85 ± 0.70% (CI: 18.48–21.22%); IC50 = 95.76 ± 10.13 μM (CI:75.90–115.61%); gamma = 1.75 ± 0.21# (CI: 1.33–2.17). Data are reported as mean value ± SE of n = 10 animals per group. Statistical significances were calculated by unpaired two‐tailed t‐test and Kruskal–Wallis test where appropriate. In vitro data are reported as mean value ± SE of five independent experiments (D, E) and raw data (with 95% CI in blue) of three independent experiments (F). ****< 0.0001; ***< 0.0003; **< 0.002.