Dietary dicarboxylic acids provide a nonstorable alternative fat source that protects mice against obesity

Abstract

Dicarboxylic fatty acids are generated in the liver and kidney in a minor pathway called fatty acid ω-oxidation. The effects of consuming dicarboxylic fatty acids as an alternative source of dietary fat have not been explored. Here, we fed dodecanedioic acid, a 12-carbon dicarboxylic (DC12), to mice at 20% of daily caloric intake for 9 weeks. DC12 increased metabolic rate, reduced body fat, reduced liver fat, and improved glucose tolerance. We observed DC12-specific breakdown products in liver, kidney, muscle, heart, and brain, indicating that oral DC12 escaped first-pass liver metabolism and was utilized by many tissues. In tissues expressing the "a" isoform of acyl-CoA oxidase-1 (ACOX1), a key peroxisomal fatty acid oxidation enzyme, DC12 was chain shortened to the TCA cycle intermediate succinyl-CoA. In tissues with low peroxisomal fatty acid oxidation capacity, DC12 was oxidized by mitochondria. In vitro, DC12 was catabolized even by adipose tissue and was not stored intracellularly. We conclude that DC12 and other dicarboxylic acids may be useful for combatting obesity and for treating metabolic disorders.

Keywords: Fatty acid oxidation; Metabolism; Mitochondria; Obesity.

Figure 1. A HFD substituted with DC₁₂ increases metabolic rate and prevents obesity.

(A and B) Male 129S1 mice (n = 6) were transitioned to a HFD or the isocaloric DC₁₂ diet at age 8 weeks. Food pellets were weighed every 2–3 days for 5 weeks to determine intake, and body weights were recorded every 2–3 days. (C and D) EchoMRI was used to assess total fat mass and lean mass after 5 weeks or 9 weeks of the special diets (n = 10). (E) Epididymal WAT (eWAT) was excised and weighed after 9 weeks on the diets. (F–I) HFD, DC₁₂, and chow-fed control mice (n = 7–8) were subjected to indirect calorimetry after 7 days on the diets. Body weight was equal at the start of indirect calorimetry (F). Whole-body respiration was measured every 30 min over a 48 hr period (G). Panels H and I are the RER and energy expenditure calculated from the data in panel G, with each separated into night versus day cycles for statistical analysis. All graphs depict means and SDs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by Student’s 2-sided t tests.

Figure 2. DC₁₂-fed mice remain glucose-sensitive.

(A–D) Male 129S1 mice (n = 8–12) were fed a HFD, the isocaloric DC₁₂ diet, or standard chow for 5 weeks and then subjected to i.p. glucose tolerance testing (GTT) after a 5-hr fast. (E) Blood glucose and insulin data at baseline (time 0 of the GTT) was used to calculate homeostatic model assessment (HOMA) values as an indicator of insulin sensitivity. (F) Oroboros high-resolution respirometry of quadriceps muscle lysates (n = 3). Base, baseline; mal, malate; pyr, pyruvate; glut, glutamate; succ, succinate; CCCP, mitochondrial uncoupler; rot, rotenone. (G–I) Acute treadmill exercise challenge to exhaustion (n = 5). Blood lactate and glucose were measured with handheld meters within 2 minutes of reaching exhaustion. All graphs represent means and SDs. Panels B, D, and E were analyzed with 1-way ANOVA and Tukey-corrected multiple comparisons and remaining panels were analyzed with 2-sided Student’s t test. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 3. Dietary DC₁₂ is excreted only in trace amounts and is not stored intracellularly.

(A) Male 129S1 mice (n = 3) were adapted to either a HFD or an isocaloric DC₁₂ diet for 5 weeks, and nighttime urine was collected for mass spectrometry to detect DCAs. (B) Male 129S1 mice (n = 4) were adapted to the DC₁₂ diet over a 5 day period, with 24-hr urine samples collected on days 0, 1, 3, and 5 for mass spectrometry to detect DCAs. (C) Similarly, fecal pellets were collected from n = 3–4 male 129S1 mice on days 0 and 7 of DC₁₂ adaptation for mass spectrometry. (D and E) Primary hepatocytes (D) or white adipose explants (E) were incubated with ¹⁴C-labeled palmitate (C₁₆) or DC₁₂ for 3 hr, washed, and extracted for lipids and FAO products (n = 5).The amount of stored versus oxidized are expressed as a percentage of the total radiolabel signal detected. (F) WAT (n = 3) blotted for the mitochondrial marker Hsp60 and Ucp1; bar graphs show densitometric analysis normalized to ponceau stain. All graphs represent means and SDs. In panel B, *P < 0.05, DC₆ versus Day 0; ^#P < 0.05, DC₈ versus Day 0. In remaining panels: **P < 0.01, ***P < 0.001, ****P < 0.0001. All were analyzed with 2-sided Student’s t test.

Figure 4. DC₁₂ is metabolized using both peroxisomes and mitochondria.

(A and B) Male 129S1 mice (n = 5) were adapted to HFD or an isocaloric DC₁₂ diet for 5 weeks. Serum was collected early during the night cycle, and tissues late in the night cycle, and they were used for mass spectrometry to detect DCAs. (C and D) Proteomics results from tissues collected after 5 weeks on the diets (n = 3), expressed as log₂ of the fold-change of DC₁₂-treated animals over HFD. Also see proteomics data in Supplemental Tables 1–6. Gold dots represent proteins that were significantly increased and blue dots are proteins that were significantly decreased (absolute log₂FC > 0.58, q < 0.05), while gray are proteins with no significant change. Black lines indicate the mean of each condition. (E) Heatmap showing the absolute levels of key peroxisomal FAO proteins across the 5 different tissues. Heatmap values are means of n = 3; ND, not detected. Asterisks indicate statistically significant pairwise differences (q < 0.01), either upregulated by DC₁₂ diet (red font) or downregulated (green font). (F–H), ¹⁴C-labeled DC₁₂ or palmitate (C₁₆) were used to probe the rates of total FAO (no etomoxir) or peroxisomal FAO (etomoxir-resistant) in whole cells. All bar graphs represent means and SDs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as determined with 2-sided Student’s t test. Liv, liver; Kid, kidney; Mus, muscle; Brn, brain; and Hrt, heart. Panel E created with BioRender.com.

Figure 5. Consumption of DC₁₂ does not cause dyslipidemia or fatty liver.

(A and B) Reactome pathway analysis of the proteomics data presented in Figure 4 revealed an upregulation of lipid synthesis pathways in liver of DC₁₂-fed mice versus HFD (A), and (B) fatty acid synthesis proteins were also upregulated in kidney and muscle. Red asterisks denote statistically significant upregulation in DC₁₂ versus HFD. See also Supplemental Tables 1–6. (C) Liver triglyceride (TAG) content of 129S1 male mice on HFD or DC₁₂ diet for 5 weeks compared with mice fed standard low-fat laboratory chow (n = 5–6). (D) Mass spectrometry was used to measure cholesterol content in mouse tissues (Liv; liver; Kid, kidney; Mus, muscle) after 5 weeks on special diets (n = 5). (E) Liver free acetate content (n = 3–4) was determined with a colorimetric kit. (F) Proteomics identified short-chain ACSS3 as being upregulated by DC₁₂ in liver. All bar graphs represent means and SDs. *P < 0.05, **P < 0.01, ***P < 0.001, as determined with 2-sided Student’s t test.

Figure 6. Dietary DC₁₂ is chain shortened to succinyl-CoA in several tissues, but circulating succinate is not increased.

(A) Immunoblotting of 20 μg of mouse tissue lysates after 7 days on HFD (HF) or DC₁₂ diets with a pan anti-succinyllysine (Suc-Lys) antibody, with Ponceau staining as loading control. Note: To visualize muscle succinylation, 40 μg protein and a longer exposure time were needed. (B) Anti-succinyllysine immunoblotting of BAT, epididymal WAT (eWAT), and inguinal WAT (iWAT). (C) Liver and kidney extracts from mice on DC₁₂ or HFD (n = 4) were used for quantitative site-level succinylomics by mass spectrometry. Peroxisomal and mitochondrial peptides were curated and plotted as log₂ fold-change (DC₁₂/HFD) to visualize the effects of DC₁₂ on succinylation in each compartment. Gold dots represent peptides with significantly increased succinylation, blue dots represent peptides with significantly decreased succinylation, and gray indicates statistical insignificance. (D) Pathway analysis of all succinylated peroxisomal proteins in liver reveals strong clustering to the fatty acid metabolism pathway; the most heavily succinylated peroxisomal proteins are depicted in E. See Supplemental Tables 7 and 8 for succinylome data sets and full protein names. (F and G) Mass spectrometry was used to measure succinate in urine and feces from male 129S1 mice during the initial 7 days of adaptation to DC₁₂ diet. (H) After chronic adaptation to the DC₁₂ diet or HFD (5 wk), mass spectrometry was used to quantify succinate in serum, urine, liver, muscle, brain, and heart. Succinate is presented as a ratio of DC₁₂: HFD, and the dashed line represents no change (ratio of 1.0). (I) The ratio of succinate to fumarate represents the substrate: product ratio for the enzyme succinate dehydrogenase, the entry point of succinate in the TCA cycle. Panel F was analyzed with 1-way ANOVA and Tukey-corrected multiple comparisons, and remaining panels were analyzed with 2-sided Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. Ser, serum; Uri, urine; Liv, liver; Kid, kidney; Mus, muscle; Brn, brain; Hrt, heart.

Figure 7. ACOX1a is required for peroxisomal generation of succinyl-CoA from DC₁₂.

(A and B) Digital droplet PCR was used for absolute quantification of total ACOX1 mRNA transcripts (A) and the 2 key isoforms ACOX1a and ACOX1b (B), expressed as copies per μg of RNA, in mouse liver (Liv), kidney (Kid), and BAT. (C–F) Characterization of recombinant human ACOX1a and ACOX1b enzyme activities with the indicated acyl-CoA substrates. Panels C and D were measured with 25 μM substrate. (G) Targeted proteomic assay employing parallel reaction monitoring was used to quantify the absolute amount of ACOX1a and ACOX1b in liver lysates of mice fed HFD versus DC₁₂ diet. (H and I) ACOX1 enzyme activity (total activity, all isoforms) detected in liver lysates of mice on HFD versus an isocaloric DC₁₂ diet. All graphs represent means and SDs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as determined with 2-sided Student’s t test.

Figure 8. DC₁₂ does not compromise peroxisomal function.

(A–D) Peroxisomes contribute to degradation of very long–chain fatty acids (VLCFA) and H₂O₂ via catalase. Mass spectrometry was used to measure the VLCFA-related metabolites lignoceric acid (C₂₄) in liver and lysophosphatidylcholine C_26:0 in serum, in mice adapted chronically (5 wk) to HFD or an isocaloric DC₁₂ diet. The amount of H₂O₂ was measured in snap-frozen liver, as was catalase enzyme activity. (E–H) Peroxisomes contribute to synthetic pathways for DHA, plasmalogens, and bile acids. Mass spectrometry was used to detect these lipid species in liver tissue (DHA) or serum (plasmalogens, bile acids). Panel F is the sum of 8 phosphatidylcholine plasmalogen species, panel G is the sum of 6 phosphoethanolamine plasmalogen species, and panel H is the sum of 11 primary C₂₄ bile acids (conjugated and unconjugated). See Supplemental Tables 9 and 10 for serum plasmalogen and bile acid data, respectively. (I) Serum alanine aminotransferase (ALT) was measured as an indicator of liver injury. All graphs represent means and SDs. Panels B and F–H were analyzed with 1-way ANOVA and Tukey-corrected multiple comparisons while remaining panels were analyzed with 2-sided Student’s t test. *P < 0.05, **P < 0.01.

Figure 9. Respiratory quotients of 12-carbon fatty acids.

(A) The respiratory quotient (RQ) of monocarboxylic C₁₂ (lauric acid) oxidized to completion by mitochondria has an RQ of 0.71. (B) DC₁₂, having 2 more oxygen molecules and 2 less hydrogens than C₁₂, has an RQ of 0.77. (C) If DC₁₂ is “predigested” by peroxisomes to acetate and succinate (above the brown lines), the oxygen requirement is reduced by 2 due to catalase reclaiming half of the oxygen used by ACOX1 in the peroxisome. This shifts the RQ up to 0.89.

Figure 10. Metabolic efficiency of DC₁₂ oxidation.

(A) The theoretical yield of ATP from oxidizing monocarboxylic C₁₂ through the mitochondrial FAO pathway and TCA cycle. Activation of fatty acids to CoA converts ATP to AMP, which is the energetic equivalent of 2 ATP (shown in red font). (B) Oxidizing DC₁₂ through mitochondria yields a remnant succinate molecule, and less acetyl-CoA, FADH, and NADH. Therefore, the ATP yield is 23% lower than for C₁₂ through the same pathway (60 versus 78). Finally, (C) oxidizing DC₁₂ through peroxisomes, then passing the succinate, acetate, and NADH into mitochondria for complete oxidation requires a much greater cost of fatty acid activation, since each acetate must be activated to CoA at the cost of 2 ATP. The result is a nearly 50% reduction in net ATP compared with C₁₂ in panel A. Image created with BioRender.com.