CPT1C in the ventromedial nucleus of the hypothalamus is necessary for brown fat thermogenesis activation in obesity

Abstract

Objective: Carnitine palmitoyltransferase 1C (CPT1C) is implicated in central regulation of energy homeostasis. Our aim was to investigate whether CPT1C in the ventromedial nucleus of the hypothalamus (VMH) is involved in the activation of brown adipose tissue (BAT) thermogenesis in the early stages of diet-induced obesity.

Methods: CPT1C KO and wild type (WT) mice were exposed to short-term high-fat (HF) diet feeding or to intracerebroventricular leptin administration and BAT thermogenesis activation was evaluated. Body weight, adiposity, food intake, and leptinemia were also assayed.

Results: Under 7 days of HF diet, WT mice showed a maximum activation peak of BAT thermogenesis that counteracted obesity development, whereas this activation was impaired in CPT1C KO mice. KO animals evidenced higher body weight, adiposity, hyperleptinemia, ER stress, and disrupted hypothalamic leptin signaling. Leptin-induced BAT thermogenesis was abolished in KO mice. These results indicate an earlier onset leptin resistance in CPT1C KO mice. Since AMPK in the VMH is crucial in the regulation of BAT thermogenesis, we analyzed if CPT1C was a downstream factor of this pathway. Genetic inactivation of AMPK within the VMH was unable to induce BAT thermogenesis and body weight loss in KO mice, indicating that CPT1C is likely downstream AMPK in the central mechanism modulating thermogenesis within the VMH. Quite opposite, the expression of CPT1C in the VMH restored the phenotype.

Conclusion: CPT1C is necessary for the activation of BAT thermogenesis driven by leptin, HF diet exposure, and AMPK inhibition within the VMH. This study underscores the importance of CPT1C in the activation of BAT thermogenesis to counteract diet-induced obesity.

Keywords: Brown adipose tissue; CPT1C; Diet-induced obesity; Hypothalamus; Thermogenesis.

Figure 1

Impaired diet-induced thermogenesis in CPT1C KO mice. (A) Representative infrared thermal images and quantification of interscapular temperature adjacent to the BAT depot of WT and CPT1C KO mice fed a standard diet (SD) or a high fat (HF) diet for 7 and 14 days. (B–D) Relative mRNA expression of the thermogenic markers UCP1 (B), PGC1α (C) and PRDM16 (D) in BAT of WT and KO mice fed SD or HF diet. (E) Protein levels of UCP1 in BAT of WT and KO fed SD or HF diet for 7 and 14 days. Data are expressed as mean ± SEM (n = 5–9). *P < 0.05, **P < 0.01, ***P < 0.001 versus WT with the same diet; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus SD within the same genotype; ⁺P < 0.05, ⁺⁺P < 0.01 versus HF 7d within the same genotype.

Figure 2

CPT1C KO mice show an earlier obesogenic phenotype compared to WT. (A and B) Body weight gain (A) and visceral WAT weight (B) of WT and KO mice fed a standard diet (SD) or a high fat (HF) diet for 7 and 14 days. (C) Representative histological H&E staining and quantification of the unilocular lipid droplets (LD) size of interscapular BAT of WT and KO mice fed a SD or a HF diet for 7 days. (D) Plasma leptin levels of WT and KO mice fat a SD or HF diet for 7 days. Data are expressed as mean ± SEM (n = 5–7). *P < 0.05, **P < 0.01, ***P < 0.001 versus WT with the same diet; ^#P < 0.05, ^##P < 0.01 versus SD within the same genotype.

Figure 3

Impaired leptin-induced thermogenesis in CPT1C KO mice. (A–C) Quantification of interscapular temperature changes adjacent to the BAT depot (iBAT) after ICV leptin treatment in WT (A) and CPT1C KO mice (B) compared with ICV vehicle. (C) Area under the curve (AUC) of iBAT temperature during 220 min. (D) Gene expression analysis of thermogenic markers in BAT of WT and KO mice after ICV leptin. Data are expressed as mean ± SEM (n = 5–8). ^#P < 0.05, ^##P < 0.01 versus Vehicle within the same genotype.

Figure 4

CPT1C KO mice show an altered expression of markers of leptin signaling and ER stress in the mediobasal hypothalamus after short-term administration of a HF diet. (A–C) Protein levels of pSTAT3 (A), mRNA levels of SOCS3 (B) and protein expression of pAMPKα, pACC, AMPK, and ACC (C) in the mediobasal hypothalamus of WT and CPT1C KO mice fed a standard diet (SD) or a high fat (HF) diet for 7 days. (D) Protein levels of pAMPKα and AMPK in the mediobasal hypothalamus of WT and CPT1C KO mice after ICV administration of leptin or vehicle. (E) mRNA levels of ER stress markers in the mediobasal hypothalamus of WT and CPT1C KO mice fed a SD or a HF diet for 7 days. Data are expressed as mean ± SEM (n = 5–7). *P < 0.05, **P < 0.01 versus WT with the same diet; ^#P < 0.05 versus SD within the same genotype; ⁺P < 0.01 versus vehicle within the same genotype.

Figure 5

Expression of CPT1C in the VMH restores short-term diet-induced response in CPT1C KO mice. (A) GFP (empty vector, EV) or CPT1C-GFP (Cpt1c)-expressing lentiviruses were microinjected in the VMH of WT and CPT1C KO mice and after 1 week, mice were fed a standard diet (SD) or a high fat (HF) diet for 7 days. (B) Injection site was confirmed by direct fluorescence of GFP in brain sections or by CPT1C expression analysis by western blot in the ventral hypothalamus. (C–E) Body weight gain (C), plasma leptin (D) and gene expression analysis of thermogenic markers in BAT of WT-EV, KO-EV, and KO expressing CPT1C (KO-Cpt1c) or CPT1CM589S (KO-Mut) fed SD or HF diet for 7 days. Data are expressed as mean ± SEM (n = 6–8). *P < 0.05 versus WT-EV-HF; ^#P < 0.05 versus WT-EV-SD; ⁺P < 0.05, ⁺⁺⁺P < 0.001 versus KO-EV-HF.

Figure 6

CPT1C KO mice show impaired AMPK-mediated effects within the VMH on body weight change, BAT thermogenesis and liver. (A) Body weight change of WT and CPT1C KO mice treated with adenoviruses encoding GFP (Empty vector, EV) or AMPK-DN in the VMH. (B–D) Representative infrared thermal images and quantification of interscapular temperature adjacent to the BAT depot (B), protein levels of UCP1 in BAT (C) and gene expression analysis of thermogenic markers in BAT of WT and KO mice treated with EV or AMPK-DN in the VMH (D). (E and F) Protein levels of the AMPK pathway (E) and TG levels in the liver of mice treated with EV or AMPK-DN in the VMH (G). Data are expressed as mean ± SEM (n = 6–7). *P < 0.05, **P < 0.01, ***P < 0.001 versus WT-EV; ^#P < 0.05, ^##P < 0.01 versus WT-AMPK-DN.