Fig 2: WB analysis of pACC1 and ACC1 in the lysates from the metabolic tissues of WT and FKO mice.(A) WB analysis of pACC1 and ACC1 in the lysates from the sWAT of WT and FKO mice fed on regular chow. Actin was used as loading control (n = 3 per group, representative of three repeats). (A, B) Quantification of the band intensity of pACC1/ACC1 ratio in (A) (n = 3 per group, each point represents a biology replicate). Data are represented as mean ± SEM, t test. (C) WB analysis of pACC1 and ACC1 in the lysates from the sWAT of WT and FKO mice fed on HFD. Actin was used as loading control (n = 3 per group, representative of three repeats). (C, D) Quantification of the band intensity of pACC1/ACC1 ratio in (C) (n = 3 each point represents a biology replicate). Data are represented as mean ± SEM, t test, *P < 0.05. (E) WB analysis of pACC1 and ACC1 in the lysates from the liver of WT and FKO mice fed on regular chow. Tubulin was used as loading control (n = 4 per group. representative of three repeats). (E, F) Quantification of the band intensity of pACC1/ACC1 ratio in (E) (n = 4 per group, each point represents a biology replicate). Data are represented as mean ± SEM, t test. (G) WB analysis of pACC1 and ACC1 in the lysates from the liver of WT and FKO mice fed on HFD. Tubulin was used as loading control (n = 4 per group, representative of three repeats). (G, H) Quantification of the band intensity of pACC1/ACC1 ratio in (G) (n = 4 per group, each point represents a biology replicate). Data are represented as mean ± SEM, t test.Source data are available for this figure.

Fig 3: CMS121 and J147 increase acetyl-CoA levels associated with inhibition of ACC1.Increases in acetyl-CoA levels are protective and associated with inhibition of ACC1 by CMS121 and J147. (A) Total levels of acetyl-CoA in rat primary neurons (n = 5/group), after treatment with 1 µM of each compound for 24 hr. Acetylation of H3 at Lys 9 (Ac-H3K9) was assessed by Western blotting and normalized to levels of total H3 in rat primary neurons (n = 3/group) (B) and mouse cortical tissue (n = 5/group) (C). (D) Protection during oxytosis in HT22 cells with CMS121 (500 nM) and J147 (50 nM) (n = 3/group). (E) Total levels of acetyl-CoA in HT22 cells (n = 3/group). Phosphorylation of ACC1 was assessed by Western blotting and normalized to levels of respective total protein in rat primary neurons (n = 3/group) (F), HT22 cells (n = 3/group) (G) and SAMP8 mouse cortical tissue (n = 5/group) (H). Scans of the blots are shown in Figure 6—figure supplement 1. (I) Fold change in the levels of different polyunsaturated fatty acids in primary neurons treated with 1 µM of each compound for 24 hr. Fold change in the levels of multiple long-chain fatty acids including major polyunsaturated fatty acids in the brains (J) and plasma (K) of old SAMP8 treated with CMS121 and J147, relative to old SAMP8. (L) Total levels of acetyl-CoA in HT22 cells treated with 5 mM glutamate alone or in the presence of 3, 10 or 30 µM TOFA, determined 8 hr after treatment (n = 3/group). (M) Protection during oxytosis in HT22 cells after inhibition of ACC1 with 3, 10 or 30 µM TOFA (n = 3/group). (N) Total levels of acetyl-CoA in HT22 cells after knock down with ACC1 siRNA, measured 48 hr after knock down (n = 3/group). Protection during oxytosis (O) and ferroptosis (P) in HT22 cells after knock down with ACC1 siRNA (n = 3/group). (Q) Protection during ferroptosis in HT22 cells with CMS121 (250 nM) and J147 (100 nM) (n = 3/group). (R) Protection during oxytosis in HT22 cells treated with sodium acetate (Ac), with survival normalized to control treatment with sodium chloride. All data are mean ± SD. One-way ANOVA followed by Tukey-Kramer post-hoc test.

Fig 4: Effects of downregulating GNAI3 expression on lipid metabolic enzymes. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of genes encoding lipid metabolic enzymes involved in fatty acid esterification, de novo lipogenesis and very low-density lipoprotein (VLDL) secretion in the livers of GNAI3KO and wild-type mice. (B) qRT-PCR analysis of ACOX, ACCa and ApoB mRNA expression levels in GNAI3 KD and control HepG2 cells. (C) Western blotting analysis of ACOX, ACCa and ApoB protein levels in GNAI3 KD and control HepG2 cells. Bar graphs show the mean±standard error of the mean (A, B) or mean±SD (C). *p<0.05 and †p<0.01, GNAI3KO/KD versus control.

Fig 5: Dapagliflozin induces autophagy and regulates fatty acid metabolism in ZDF rats and PA-stimulated LO2 and HepG2 cells. (A) IHC staining detected the expression of LC3B, Beclin-1 and p62 in hepatic tissue. Scale bars: 20 µm. Data are expressed as the means ± SEM of five animals per group. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the ZDF rat group. (B) Western blot analysis evaluated the protein expression levels of LC3B, Beclin-1, and p62 in rats. (C) Western blot analysis detected the expression of ACC1, p-ACC1, and ACOX1 in the livers of rats. (D, E) LC3B immunofluorescence staining showed the endogenous LC3B level of LO2 and HepG2 cells. (F, G) Western blot analysis demonstrated the expression of LC3B, Beclin-1, and p62 in LO2 and HepG2 cells. (H, I) Western blot analysis detected the protein expression of ACC1, p-ACC1 and ACOX1 in cells. (J, K) LO2 and HepG2 cells were treated with 0.3 mM PA, 20 µM dapagliflozin and 20 µM chloroquine (CQ) for 24 h. The cells were stained with Oil Red O and intracellular TG was quantitatively analyzed. Scale bars: 20 µm. Data are expressed as the means ± SEM from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.