Hepatic Fatty Acid Oxidation Restrains Systemic Catabolism during Starvation

Abstract

The liver is critical for maintaining systemic energy balance during starvation. To understand the role of hepatic fatty acid β-oxidation on this process, we generated mice with a liver-specific knockout of carnitine palmitoyltransferase 2 (Cpt2(L-/-)), an obligate step in mitochondrial long-chain fatty acid β-oxidation. Fasting induced hepatic steatosis and serum dyslipidemia with an absence of circulating ketones, while blood glucose remained normal. Systemic energy homeostasis was largely maintained in fasting Cpt2(L-/-) mice by adaptations in hepatic and systemic oxidative gene expression mediated in part by Pparα target genes including procatabolic hepatokines Fgf21, Gdf15, and Igfbp1. Feeding a ketogenic diet to Cpt2(L-/-) mice resulted in severe hepatomegaly, liver damage, and death with a complete absence of adipose triglyceride stores. These data show that hepatic fatty acid oxidation is not required for survival during acute food deprivation but essential for constraining adipocyte lipolysis and regulating systemic catabolism when glucose is limiting.

Figure 1. Characterization of mice with a liver specific KO of CPT2

(A) Western blot for CPT2 in liver of Cpt2^lox/lox and Cpt2^L−/− mice. (B) mRNA for Cpt2 across different tissues (n=6). (C) Oxidation of 1-¹⁴C-oleic acid and 1-¹⁴C-Lignoceric acid to ¹⁴CO₂ in liver slices of Cpt2^lox/lox and Cpt2^L−/− mice (n=5). (D) Body weights of Cpt2^lox/lox and Cpt2^L−/− male and female mice fed a normal chow diet (males, n=14-23; females, n=8-12). Data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Figure 2. Liver and systemic deficits in fed and 24hr fasted Cpt2^L−/− mice

(A) Serum metabolites in Cpt2^lox/lox and Cpt2^L−/− mice (n=6). (B) Energy expenditure and respiratory exchange ratio of Cpt2^lox/lox and Cpt2^L−/− mice under fed, fast and refed conditions (males, n=5-7). (C) Total fat and lean mass of Cpt2^lox/lox and Cpt2^L−/− male mice (n=5-7). (D) Wet weights of fed or 24 hour fasted iWAT, gWAT, and liver for Cpt2^lox/lox and Cpt2^L−/− mice (n=6-10). (E) Gross and histological morphology of livers from 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice. Scale bar equals 100 μM. (F) Triglyceride levels from liver homogenates of fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice (n=5). (G) Liver damage measured by serum ALT activity of fed and 24h fasted Cpt2^lox/lox and Cpt2^L−/− mice (n=5). (H) TBARS assay measuring lipid peroxidation from liver of fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice (n=5). Data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Figure 3. Loss of hepatic fatty acid oxidation induces expression of fatty acid oxidative genes

(A) Gene expression of fatty acid oxidation genes in liver of fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice (n=6). (B) Western blots of proteins in fatty acid metabolism. Composite of 8 blots. All blots were normalized to Hsc70 (Figure S2). (C) Liver mRNA (n=6) of Fgf21, Gdf15 and Igfbp1 in fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice. (D) Serum concentrations (n=8) of Fgf21, Gdf15 and Igfbp1 in fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice. Data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Figure 4. Loss of hepatic fatty acid oxidation results in compensation from the kidney, muscle and adipose tissue

(A) Gene expression of fatty acid oxidation genes in the kidney of fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice (n=6). (B) Western blots of proteins in fatty acid metabolism. Composite of 5 blots. All blots were normalized to Hsc70 (Figure S3). (C) Gross kidney morphology in 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice. (D) Kidney wet weight of fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice (n=6-10). (E) Kidney TAG content of fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice (n=5). (F) iBAT gene expression of fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice (n=6). (G) iWAT gene expression of fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice (n=6). (H) gWAT adiponectin mRNA (n=6) and Adiponectin serum concentration (n=8) of fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice. (I) Gene expression of fatty acid oxidation genes in the gastrocnemius muscle of fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice (n=6). (J) Gastrocnemius muscle mRNA of Fgf21 in fed and 24 hr fasted Cpt2^lox/lox and Cpt2^L−/− mice (n=6). Data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Figure 5. A ketogenic diet results in hypoglycemia, a depletion of adipose triglyceride and eventually lethality in Cpt2^L−/− mice

(A) Body weights of Cpt2^lox/lox and Cpt2^L−/− mice fed a normal chow or ketogenic diet for 6 days (normal chow, n=6-10; ketogenic diet, n=5-6). (B) Wet weight of liver from Cpt2^lox/lox and Cpt2^L−/− mice fed a ketogenic diet for 6 days (n=5-6). (C) Liver damage measured by serum ALT activity of Cpt2^lox/lox and Cpt2^L−/− mice fed a ketogenic diet (n=5). (D) Serum metabolites in Cpt2^lox/lox and Cpt2^L−/− mice after a 6-day ketogenic diet (n=5-6). (E) Gross morphology of Cpt2^lox/lox and Cpt2^L−/− mice fed a ketogenic diet. (F) Gene expression of fatty acid oxidation genes in the liver of Cpt2^lox/lox and Cpt2^L−/− mice fed a ketogenic diet (n=6). (G) Serum concentrations of Fgf21, Gdf15 and Igfbp1 of Cpt2^lox/lox and Cpt2^L−/− mice fed a ketogenic diet (n=6). Data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Figure 6. Time course of body weight, blood glucose and acylcarnitines in Cpt2^L−/− mice fed a ketogenic diet

(A) Blood glucose of Cpt2^lox/lox and Cpt2^L−/− mice during a 4-day ketogenic diet (n=5). (B) Body weight of Cpt2^lox/lox and Cpt2^L−/− mice during a 4-day ketogenic diet (n=5). (C) Liver and blood L-carnitine of Cpt2^lox/lox and Cpt2^L−/− mice following a 4-day ketogenic diet (n=5). (D) Total blood acylcarnitines and acetylcarnitine of Cpt2^lox/lox and Cpt2^L−/− mice following a 4-day ketogenic diet (n=5). (E) Daily blood long chain (C18:0, C18:1) acylcarnitines of Cpt2^lox/lox and Cpt2^L−/− mice during a 4-day ketogenic diet (n=5). (F) Total liver acylcarnitines and acetylcarnitine of Cpt2^lox/lox and Cpt2^L−/− mice following a 4-day ketogenic diet (n=4-5). (G) Total liver long chain (C16:0, C18:0, C18:1) acylcarnitines and acetylcarnitine of Cpt2^lox/lox and Cpt2^L−/− mice following a 4-day ketogenic diet (n=4-5). Data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.