Adipose fatty acid oxidation is required for thermogenesis and potentiates oxidative stress-induced inflammation

Abstract

To understand the contribution of adipose tissue fatty acid oxidation to whole-body metabolism, we generated mice with an adipose-specific knockout of carnitine palmitoyltransferase 2 (CPT2(A-/-)), an obligate step in mitochondrial long-chain fatty acid oxidation. CPT2(A-/-) mice became hypothermic after an acute cold challenge, and CPT2(A-/-) brown adipose tissue (BAT) failed to upregulate thermogenic genes in response to agonist-induced stimulation. The adipose-specific loss of CPT2 resulted in diet-dependent changes in adiposity but did not result in changes in body weight on low- or high-fat diets. Additionally, CPT2(A-/-) mice had suppressed high-fat diet-induced oxidative stress and inflammation in visceral white adipose tissue (WAT); however, high-fat diet-induced glucose intolerance was not improved. These data show that fatty acid oxidation is required for cold-induced thermogenesis in BAT and high-fat diet-induced oxidative stress and inflammation in WAT.

Figure 1. Generation of mice with an adipose specific KO of CPT2

(A) Gene targeting strategy for the Cpt2 gene. Triangles represent LoxP sites. (B) mRNA for Cpt2 in adipose depots and liver of control and CPT2^A−/− mice (n=8). (C) Western blot for CPT2 in BAT of control, CPT2^A−/+ and CPT2^A−/− mice. (D) Oxidation of 1-¹⁴C-Oleic acid to ¹⁴CO₂ in control and CPT2^A−/− adipose depot explants (n=5). (E) Oxidation of 1-¹⁴C-Oleic acid or 1-¹⁴C-Lignoceric acid to ¹⁴CO₂ in control and CPT2 KO MEFs (n=5). (F) Acyl-Carnitine profile of iWAT in control and CPT2^A−/− mice (n=8). Data are expressed as means +/− SEM. *p<0.05, **p<0.001, N.S. not significant. Open bars represent control and black bars represent loss of CPT2.

Figure 2. Adipose fatty acid oxidation is required for acute cold induced thermogenesis

(A) Body temperature of control and CPT2^A−/− mice subjected to a 3hr cold challenge (n=10–13). (B) Body weights of 12 week old female control and CPT2^A−/− mice (n=22–26). (C) Gross morphology of control and CPT2^A−/− BAT after 2hr of cold exposure. (D) H&E stained sections of BAT from control and CPT2^A−/− mice at 21°C and 3hr at 4°C. Scale bar is 100μM. (E) Triglyceride content of BAT of control and CPT2^A−/− mice after 3hr at 4°C (n=5). (F) Serum metabolites in control and CPT2^A−/− mice at 21°C and 3hr at 4°C (n=8). Data are expressed as means +/− SEM. *p<0.001. Open bars represent control and black bars represent loss of CPT2.

Figure 3. Adipose fatty acid oxidation is required for agonist induced thermogenic gene expression and mitochondrial homeostasis

(A) mRNA expression of fatty acid oxidative genes in BAT of control and CPT2^A−/− mice at 21°C or after 3hr at 4°C (n=8). (B) mRNA expression of Ucp1, Pgc1α, and Dio2 in BAT of control and CPT2^A−/− mice at 21°C (n=8), after 3hr at 4°C (n=8), or 3hr after injection with 10mg/kg CL-316243 (n=5). (C) mRNA expression of Ucp1 in BAT explants treated with 10uM CL-316243, isoproterenol, or forskolin (n=5). (D) Western blot for PKA phosphorylated substrates in BAT of control and CPT2^A−/− mice treated with 10mg/kg CL-316243 for 30min in vivo. (E) Western blot for P-CREB (Ser-133) in BAT of control and CPT2^A−/− mice treated with 10mg/kg CL-316243 for 30min in vivo. (F) Body temperature of control and CPT2^A−/− mice acclimatized to 15°C and subjected to a 4hr cold challenge at 4°C (n=5). (G) mRNA expression of Ucp1, Pgc1α, and Dio2 in BAT of 15°C acclimatized control and CPT2^A−/− mice after a 4hr cold challenge (n=5). (H) mRNA expression of Ucp1, Pgc1α, and Dio2 in BAT of control and CPT2^A−/− mice acclimatized to 30°C and injected with vehicle or 10mg/kg CL-316243 for 3hrs (n=4–5). (I) Western blot of mitochondrial proteins in BAT of control and CPT2^A−/− mice at 21°C or after 3hr at 4°C. (J) Mitochondrial DNA content of BAT and gWAT from control and CPT2^A−/− mice (n=10–12). Data are expressed as means +/− SEM. * p<0.01, **p<0.05. Open bars represent control and black bars represent loss of CPT2.

Figure 4. Contribution of adipose fatty acid oxidation to energy expenditure

(A) VO2 consumption of control and CPT2^A−/− male mice treated with 10mg/kg CL-316243. (B) Food intake of control and CPT2^A−/− mice. (C) Water intake of control and CPT2^A−/− mice under ad libitum and fasting in under dark and light cycles. (D) Respiratory Exchange Ratio of control and CPT2^A−/− mice under ad libitum and fasting in under dark and light cycles. (E) Energy expenditure of control and CPT2^A−/− mice under ad libitum and fasting in under dark and light cycles. (F) Ambulation rates of control and CPT2^A−/− mice under ad libitum and fasting in under dark and light cycles (n=10–14). Data are expressed as means +/− SEM. *p<0.05. Open bars represent control and black bars represent loss of CPT2.

Figure 5. The loss of adipose fatty acid oxidation affects diet dependent adiposity but not body weight

(A) Body weights of control and CPT2^A−/− male mice fed a low or high fat diet (n=13–18). (B) H&E stained sections of gWAT from control and CPT2^A−/− mice fed low or high fat diets. Scale bar is 250μM. (C) Body compositions measured by EchoMRI for control and CPT2^A−/− mice fed low or high fat diets (n=13–18). (D) Wet weights of iWAT and gWAT unilateral depots for control and CPT2^A−/− mice fed low or high fat diets (n=13–18). (E) Serum metabolites in control and CPT2^A−/− mice fed low or high fat diets (n=8). Data are expressed as means +/− SEM. **p<0.01, *p<0.05. Open bars represent control and black bars represent loss of CPT2.

Figure 6. The loss of fatty acid oxidation alters carbohydrate metabolic flux

(A) De novo lipogenesis of control and CPT2^A−/− liver, gWAT and BAT from a 1hr injection of ³H-Acetate normalized to tissue wet weight. (n=4–5). (B) De novo lipogenesis of control and CPT2^A−/− MEFs from ³H-Acetate or 2-¹⁴C-Pyruvate normalized to protein concentration. (n=6). (F) Substrate oxidation of control and CPT2^A−/− MEFs from 2-¹⁴C-Pyruvate or U-¹⁴C-Glucose normalized to protein concentration. (n=5). Data are expressed as means +/− SEM. *p<0.05. Open bars represent control and black bars represent loss of CPT2.

Figure 7. Adipose fatty acid oxidation potentiates high fat induced oxidative stress and inflammation

(A) qRT-PCR of oxidative stress genes from gWAT of control and CPT2^A−/− mice fed low or high fat diets (n=8). (B) qRT-PCR of adipokines from gWAT of control and CPT2^A−/− mice fed low or high fat diets (n=8). (C) qRT-PCR of inflammatory genes from gWAT of control and CPT2^A−/− mice fed low or high fat diets (n=8). (D) TBARS assay from gWAT and serum of control and CPT2^A−/− mice fed low or high fat diets (n=5). (E) ipGTT and ipITT including area under the curve and area above the curve, respectively for control and CPT2^A−/− mice fed a low fat diet (n=9). (F) ipGTT and ipITT including area under the curve and area above the curve, respectively for control and CPT2^A−/− mice fed a high fat diet (n=13–18). Data are expressed as means +/− SEM. **p<0.005, *p<0.05