Carnitine palmitoyltransferase 1 facilitates fatty acid oxidation in a non-cell-autonomous manner

Abstract

Mitochondrial fatty acid oxidation is facilitated by the combined activities of carnitine palmitoyltransferase 1 (Cpt1) and Cpt2, which generate and utilize acylcarnitines, respectively. We compare the response of mice with liver-specific deficiencies in the liver-enriched Cpt1a or the ubiquitously expressed Cpt2 and discover that they display unique metabolic, physiological, and molecular phenotypes. The loss of Cpt1a or Cpt2 results in the induction of the muscle-enriched isoenzyme Cpt1b in hepatocytes in a Pparα-dependent manner. However, hepatic Cpt1b does not contribute substantively to hepatic fatty acid oxidation when Cpt1a is absent. Liver-specific double knockout of Cpt1a and Cpt1b or Cpt2 eliminates the mitochondrial oxidation of non-esterified fatty acids. However, Cpt1a/Cpt1b double knockout mice retain fatty acid oxidation by utilizing extracellular long-chain acylcarnitines that are dependent on Cpt2. These data demonstrate the non-cell-autonomous intercellular metabolism of fatty acids in hepatocytes.

Keywords: CP: Metabolism; Cpt1; Cpt2; acylcarnitine; biochemistry; fasting; liver; metabolism.

Figure 1.. Deletions of Cpt1a and Cpt2 have unique transcriptional responses and physiology despite lipid accumulation.

(A) Schematic depicting localization and function of Cpt1 and Cpt2. (B) Western blot for Cpt1a and Cpt2 in liver of control, Cpt1a^L−/−, and Cpt2^L−/− mice. (C) Wet tissue weights of control, Cpt1a^L−/−, and Cpt2^L−/− mice (n= 5–10). (D) Serum metabolites in control, Cpt1a^L−/−, and Cpt2^L−/− mice (n = 6). (E) Gross physiology, H&E stain (scale bar, 50 μm), and BODIPY stain of neutral lipid in control, Cpt1a^L−/−, and Cpt2^L−/− mice (scale bar, 50 μm). (F) Thin-layer chromatography plate with different lipid fractions shown and triglyceride quantification in control, Cpt1a^L−/−, and Cpt2^L−/− mice (n = 6). (G) Principal-component analysis (PCA) of RNA sequencing data between control, Cpt1a^L−/−, and Cpt2^L−/− fasted livers (n = 4). (H) Volcano plot of significantly different genes in Cpt1a^L−/− livers compared to Cpt2^L−/− shown in red points. (I) Follow-up qPCR of fgf21, gdf15, igfbp1, atf3, and cd68 in control, Cpt1a^L−/−, and Cpt2^L−/− livers. Data are expressed as mean ± SEM. Single letter denotes p < 0.05 between genotypes. *p < 0.05, **p < 0.01, and ***p < 0.001, by one-way ANOVA with Bonferroni post hoc correction.

Figure 2.. Pparα is sufficient and required for the upregulation of cpt1b.

(A) qPCR analysis of cpt1b across control, Cpt1a^L−/−, Cpt2^L−/−, Cpt1a/2^L−/−, and CptTKO^L−/− fasted livers (n = 6). (B) qPCR analysis of cpt1b in wild-type primary hepatocytes treated with 10 μM WY-14643, a Pparα agonist (n = 3). (C) qPCR analysis of cpt1b across Ppara^fl/flCpt2^fl/fl, Cpt2^fl/fl, and Ppara^fl/fl mice injected with either EGFP or iCre (n = 6). (D) qPCR analysis of Pparα targets fgf21, gdf15, pdk4, and acox1 across Ppara^fl/flCpt2^fl/fl, Cpt2^fl/fl, and Ppara^fl/fl mice injected with either EGFP or iCre (n = 6). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, by one-way ANOVA with Bonferroni post hoc correction.

Figure 3.. Cpt1b does not contribute significantly to the hepatic fasting response.

(A) qPCR analysis of cpt1a, cpt1b, and cpt2 across WT, Cpt1a/b^L−/−, Cpt2^L−/−, and CptTKO^L−/− fasted livers (n = 6). (B) Gross anatomy of Cpt1a^fl/fl;Cpt1b^fl/fl;Cpt2^fl/fl and CptTKO^L−/− livers following a 24 h fast. (C) Representative H&E staining of control, Cpt1a/b^L−/−, Cpt2^L−/−, and CptTKO^L−/− fasted mouse livers (scale bar, 50 μm). (D) Total body weights and wet tissue weights of control, Cpt1a/b^L−/−, Cpt2^L−/−, and CptTKO^L−/− mice following a 24 h fast (n= 7–11). (E) Serum metabolites in control, Cpt1a/b^L−/−, Cpt2^L−/−, and CptTKO^L−/− mice following a 24 h fast (n = 6). (F) PCA of RNA sequencing data showing control, Cpt1a/b^L−/−, Cpt2^L−/−, and CptTKO^L−/− 24 h fasted hepatic transcriptome (n = 4). (G) Volcano plot of significantly changed genes (red) in Cpt2^L−/− compared to Cpt1ab^L−/− fasted livers. Data are expressed as mean ± SEM. Single letter denotes p < 0.05 between genotypes. *p < 0.05, **p < 0.01, and ***p < 0.001, by one-way ANOVA with Bonferroni post hoc correction.

Figure 4.. Exogenous acylcarnitine uptake masks the loss of hepatic acylcarnitine generation.

(A) Oxidation of exogenous 1-¹⁴C oleic acid or 1-¹⁴C palmitoylcarnitine to CO₂ in primary hepatocytes isolated from control, Cpt1a/b^L−/−, and Cpt2^L−/− mice (n = 6). (B) Lysine acetylation western blot in fasted livers of control, Cpt1a/b^L−/−, Cpt2^L−/−, and CptTKO^L−/− mice (n = 3). (C) qPCR analysis of fgf21 and gdf15 mRNA in liver and Fgf21 and Gdf15 protein in serum of fasted control, Cpt1a/b^L−/−, Cpt2^L−/−, and CptTKO^L−/− male and female mice (n = 6). (D) Acylcarnitine analysis of short-chain and long-chain acylcarnitine species in fasted control, Cpt1a/b^L−/−, Cpt2^L−/−, and CptTKO^L−/− livers (n = 3). Data are expressed as mean ± SEM. Single letter denotes p < 0.05 between genotypes. *p < 0.05, **p < 0.01, and ***p < 0.001, by one-way ANOVA with Bonferroni post hoc correction.