Fig 2: Targeting lipid droplet homeostasis has various effects on F. tularensis intracellular growth. (A) Lipid droplet (LD) homeostasis pathways. ACS, acyl CoA synthetase; DGAT, diacylglycerol acyltransferase; ATGL, adipose triglyceride lipase; ACC, acetyl-CoA carboxylase; FA, fatty acid. TAG, triacylglycerol; DAG, diacylglycerol; MAG, monoacylglycerol; MAGL, monoacylglycerol lipase. (B) Growth kinetics of WT MEFs treated with 30 µM atglistatin, 5 µM triacsin C, 10 µM T863, or DMSO as a control and infected with WT F. tularensis Schu S4 harboring a luciferase plasmid (LUX). Luminescence (RLU) was measured over 24 h. Data points are the means ± SD and represent results from 3 biological replicates performed in triplicate. (C) Growth kinetics of BMDMs treated with 30 µM atglistatin, 5 µM triacsin C, 10 µM T863, or DMSO as a control and infected with WT F. tularensis Schu S4 harboring a luciferase plasmid (LUX). Luminescence (RLU) was measured over 24 h. Data points are the means ± SD and represent the results of 3 biological replicates performed in triplicate.

Fig 3: Mice with acute deletion of OGT in VMH neurons show reduced energy expenditure and adipocyte hypertrophy associated with reduced lipolysis of white adipose tissues.(A to D) Metabolic cage analysis of food intake (A), physical activity (B), energy expenditure (C), and RER (D) of GFP and Cre mice 2 weeks after stereotaxic virus injection before body weight divergence. (E) H&E staining of scWAT, pgWAT, and rpWAT of CTL and VOK mice after 6 hours of fasting. Scale bar, 100 µm. (F) Adipocyte sizes in different white adipose tissues of CTL and VOK mice. Individual dots represent individual adipocytes from three GFP and four Cre mice. (G) Serum nonesterified fatty acid (NEFA) levels of GFP and Cre mice fed ad libitum and after 15 hours of fasting. (H and I) Western blot of acetyl-CoA carboxylase (ACC), p-ACC (S79), ATGL, p-ATGL (S406), HSL, p-HSL (S563), and p-HSL (S565) in scWAT (H) and pgWAT (I) of GFP and Cre mice after 6 hours of fasting. Densitometric quantifications for the expression levels of ATGL, p-ATGL (S406), HSL, and p-HSL (S563) are shown on the right. (J and K) Western blot of p-PKA substrate in scWAT (J) and pgWAT (K) of GFP and Cre mice after 6 hours of fasting. Densitometric quantification is shown below. GFP: n = 4 and Cre: n = 4 for metabolic cage analysis. GFP: n = 8 to 10 and Cre: n = 8 to 10 for serum NEFA. Data are shown as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by unpaired Student’s t test. A.U., arbitrary units.

Fig 4: Seven-day HFD feeding impairs insulin-stimulated insulin signaling cascade in WAT associated with increases in plasma membrane sn-1,2-DAGs and PKCe translocation.(A–C) Insulin-stimulated phosphorylation of Insr, Akt, and PDE3B in WAT. (D and E) WAT cAMP and PKA activity during the hyperinsulinemic-euglycemic clamp. (F–H) Insulin-stimulated phosphorylation of HSL, perilipin, and ATGL. (I) WAT PKCe membrane/cytosol ratio. (J) Separation of 5 subcellular compartments in WAT: plasma membrane (PM), mitochondria (Mito), ER, cytosol (C), and lipid droplet (LD). (K) WAT sn-1,2-DAGs in 5 compartments. In A–H, rats (after overnight fasting) were under hyperinsulinemic-euglycemic clamp conditions. Data are the mean ± SEM of n = 5–10 per group. In I–K, rats were under 6-hour fasting basal condition; data are the mean ± SEM of n = 4–5 per group. In all panels, groups are compared by 2-tailed unpaired Student’s t test. *P < 0.05, **P < 0.01.

Fig 5: cAMP/PKA signal pathway was essential for effects of ColXV on adipocyte differentiation and lipolysisAdipocytes were pre-infected with Ad-Col15a1 and sh-Col15a1 (Ad-GFP as control) for 72 h. Part of those cells were incubated with 10 µM Forskolin for 1 h, then cAMP level (A) protein level of CREB, p-PKA/PKA (B) and C/EBPß, FABP4, ATGL, p-ATGLSer406 (D) were detected (n = 6). Another part of those cells were treated with 10 µM H89 for 2 h, followed by cAMP level (C) protein level of CREB, p-PKA and PKA (E) and C/EBPß, FABP4, ATGL, p-ATGLSer406 (F) evaluation (n = 6). Data represent the mean ± SEM of three independent experiments. *#P < 0.05, **##P < 0.01.