Fig 1.
Nutritional perturbation in mice.
(A) Body weights of male C57Bl/6J mice for 12 weeks on either control diet (CD), high-fat diet (HFD), or ketogenic diet (KD) n = 15–30. (B) Serum non-esterified free fatty acids (NEFA), (C) beta-hydroxybutyrate (β-OH), and (D) blood glucose in CD, HFD, KD, Fasted (Fast), Fasting-Refed (FR), and cold exposed mice, n = 8–10. (E) Whole body, (F) liver, (G) gonadal white adipose, and (H) inguinal white adipose weight in CD, HFD, KD, Fast, FR, and cold exposed mice, n = 10–15. Data represent mean ± SEM, * represents p≤0.05 by Student’s t-test relative to the CD group. Significant differences among group means are represented by letters and were determined by Tukey multiple comparison tests (p<0.05) after one-way ANOVA.
Fig 2.
Relative tissue distribution of the acyl-CoA thioesterases.
Percent tissue distribution of each enzyme was determined by qPCR in relation to the sum of its expression across all tissues and all dietary conditions for the liver (Liv), heart (Hrt), kidney (Kid), gonadal white adipose (Gon), inguinal white adipose (Ing), soleus muscle (Sol), plantaris muscle (Plant), duodenum (Gut), whole brain (Brain), and brown adipose tissue (BAT), n = 20–45. Data represent mean percent ± SEM.
Fig 3.
Relative tissue distribution of the acyl-CoA synthetases.
Percent distribution of each enzyme was determined by qPCR in relation to the sum of its expression across all conditions for the liver (Liv), heart (Hrt), kidney (Kid), gonadal white adipose (Gon), inguinal white adipose (Ing), soleus muscle (Sol), plantaris muscle (Plant), duodenum (Gut), whole brain (Brain), and brown adipose tissue (BAT), n = 20–45. Data represent mean percent ± SEM.
Fig 4.
Nutritional modulation of ACOT enzymes.
Fold-change of tissue mRNA abundance for each gene, relative to control diet, for high-fat diet (HFD), ketogenic diet (KD), overnight fasted (Fast), overnight fasted followed by 12-hour refeeding (FR), or cold exposed (Cold) mice (n = 6–8). ND indicates not detectable. Significant differences between CD and all other groups represented in yellow and were determined by Tukey multiple comparison tests (p<0.05) after one-way ANOVA, except for cold treatment which was analyzed by Student’s t-test. Complete statistical analysis via one-way ANOVA is provided in S1 Fig.
Fig 5.
Nutritional modulation of ACS enzymes.
Fold-change of tissue mRNA abundance for each gene, relative to control diet, for high-fat diet (HFD), ketogenic diet (KD), overnight fasted (Fast), overnight fasted followed by 12-hour refeeding (FR), or cold exposed (Cold) mice (n = 6–8). ND indicates not detectable. Significant differences between CD and all other groups represented in yellow and were determined by Tukey multiple comparison tests (p<0.05) after one-way ANOVA except for cold treatment which was analyzed by Student’s t-test. Complete statistical analysis via one-way ANOVA is provided in S2 Fig.
Fig 6.
Tissue-specific posttranscriptional regulation of Acot1 and Acot7.
Protein abundance for (A) Acot1 and (F) Acot7 across tissues expressed as percent of total protein visualized, n = 3. Gene mRNA and protein abundance across dietary conditions, relative to control diet group, in (B,G) liver, (C,H) heart, (D,I) gonadal white adipose tissues (gWAT), and (E,J) inguinal white adipose tissue (iWAT) for Acot1 and Acot7, respectively, n = 5–6. Significant differences were determined by Tukey multiple comparison tests (p<0.05) after one-way ANOVA. Images of blots are provided in S3 Fig.
Fig 7.
Development of a transgenic mouse model with a conditional tissue-specific and cytoplasmically targeted long-chain acyl-CoA thioesterase.
(A) Transgenic construct schematic and representative western blot confirming Acot7HA-FLAG overexpression in liver. (B) Thioesterase activity for oleoyl-CoA in liver lysate from control and Acot7HA-Liv transgenic mice, n = 5–7. Overnight fasted control and Acot7HA-Liv liver slice rates of (C) 14C-oleate oxidation, (D) 14C-oleate incorporation into complex lipids, and (E) 3H-acetate incorporation into lipids, n = 5–7. Data represent mean ± SEM, * represent p≤0.05 by Student’s t-test.
Fig 8.
Doubling of hepatic cytoplasmic long-chain acyl-CoA thioesterase activity does not alter liver fatty acid metabolism.
Control and Acot7HA-Liv (A) weight gain, (B) response to glucose tolerance test, (C) liver weight, and (D) liver triacylglycerol (TAG) in response to high-fat diet feeding for 11 weeks, n = 8–12. Control and Acot7HA-Liv (E) liver weight, (F) liver TAG, (G) serum non-esterified fatty acids (NEFA), (H) serum β-hydroxybutyrate, (I) blood glucose, and (J) liver mRNA abundance of gluconeogenic genes in response to overnight fasting (18 hours), n = 7–11. Data represent mean ± SEM.
Fig 9.
Increased adipocyte cytoplasmic long-chain acyl-CoA thioesterase activity inhibits cold-induced thermogenesis but does not protect against diet-induced obesity.
A) Representative Acot7 western blot and (B) thioesterase activity for oleoyl-CoA in control and Acot7HA-Adi gonadal adipose (gWAT), inguinal adipose (iWAT), and brown adipose (BAT), n = 3–12. Control and Acot7HA-Adi (C) weight gain, (D) response to glucose tolerance test, and (E) inguinal and gonadal adipose weight in response to high-fat diet feeding for 11 weeks, n = 3–12. Control and Acot7HA-Adi (F) body temperature and (G) mRNA abundance of adrenergic genes in brown adipose in response to acute 4 hour cold (4°C) exposure, n = 8–10. Data represent mean ± SEM, * represent p≤0.05 by Student’s t-test.