Fig 2: Deletion of p53 suppresses adipocyte lipid metabolism through the down-regulation of lysosomal acid lipase.(A) Long-term lipolysis activities were measured in Iso-stimulated p53-knockout (KO) and wild-type (WT) control adipocytes. (B) LAL mRNA expression levels were examined in the samples from the end time point (36 hr) of A (C) Protein expression levels of LAL, ATGL, and actin were examined by western blots in control, LAL, ATGL, and LAL/ATGL double RNAi knockdown cells. (D) 3T3-L1 adipocytes from C were cultured with or without ISO stimulation (in PBS +1% BSA) for 18 hr and the concentrations of fatty acid in the medium were measured. (*p<0.05). (E) LAL was overexpressed in WT and p53–KO 3T3-L1 adipocytes, and (F) long-term lipolysis (18 hr) activities were measured by determining FFA levels. (*p<0.05, **p<0.01). Figure 4—source data 1.An Excel sheet with numerical quantification data.

Fig 3: A working model for non-canonical p53 pathway-regulated adipocyte metabolic flexibility.Nutrient starvation causes an AMPK-dependent p53 non-canonical pathway activation, which leads to the upregulation of LAL-mediated lysosome acid lipolysis. This pathway plays key role for adipocyte oscillating between glycolysis and lipolysis under feeding and fasting states. In insulin resistance, p53 is found to be upregulated, while p53 downregulation is associated with insulin sensitivity.

Fig 4: Deletion of p53 increases adipocyte glucose metabolism.(A) In vitro working model for inducible p53 knockout (KO) in 3T3-L1 adipocytes. (B) p53-knockout (KO) and wild-type (WT) cells were cultured in no-glucose DMEM with 0.1% FBS for overnight, then glucose was added to media to a final concentration of 5 mM. 10–20 µl culture media were sampled every 12 hr. Glucose concentrations were measured using commercially available kits. (C–D) Glycolytic and mitochondrial functions were measured by Seahorse. Relative NADPH levels (E) and G6DP mRNA expression levels (F) were examined. (G) Lactate levels were measured similarly as in (B). (*p<0.05). Figure 2—source data 1.An Excel sheet with numerical quantification data.

Fig 5: Metabolic regulation of p53 is mediated through AMPK-dependent phosphorylation.(A–B) Nuclear fraction was prepared from control (C, High-glucose DMEM with 10% FBS), fasted (F, PBS with 1% BSA and ISO for 6 hr), AICAR (AI), or compound C treated before fasting (Inhi+F), 3T3-L1 adipocytes, following by immunoprecipitation and western blots. (C) Quantification of B. (D) LAL mRNA expression levels were examined by RT-PCR in the cells from B. (E) The effect of metformin on fasting-induced p53 phosphorylation was determined by immunoprecipitation and western blots. (F–G) Effects of metformin on glucose consumption and lipolysis in cultured 3T3-L1 WT and p53-KO adipocytes were determined. (*p<0.05). Figure 6—source data 1.An Excel sheet with numerical quantification data.