Fig 2: Effects of PPH on activating AMPK signaling pathway in WAT. Western blotting analyses of pAMPK, pHSL, and pACC protein expression in WAT (A); quantification of the pAMPK/AMPK, pHSL/HSL, and pACC/ACC ratios (B). *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 3: The Role of ATGL and HSL in IL-6 and β-adrenergic Adipocyte Signaling and Lipolysis. (A–D) Pre-adipocytes in a 12-well format were infected with lentivirus containing Cas9 and sgGFP (black), sgPnpla2 (red), or sgLipe (blue) followed by differentiation as described in Methods. On day 8, differentiated adipocytes were treated in a final volume of 1 ml of medium D with 3 µl DMSO and 3 µl PBS containing the indicated concentration of isoproterenol (A, B), IL-6 (A, C), or LIF (A, D). After incubation for 24 hours, medium was collected and processed to measure concentrations of glycerol (left panel) or NEFA (right panel) and adipocytes were harvested and processed for immunoblot analysis (A) with the indicated antibodies as described in Methods. Glycerol levels are shown relative to the average 300 nM isoproterenol condition (B, 578 µM), 3 nM IL-6 condition (C, 140 µM), or 1 nM LIF condition (D, 86 µM) of the sgGFP-treated adipocytes. NEFA levels are shown relative to the average 300 nM isoproterenol condition (B, 123 µM), 3 nM IL-6 condition (C, 57 µM), or 1 nM LIF condition (D, 27 µM) of the sgGFP-treated adipocytes. Data is shown as mean ± SEM of three wells. P value calculated by using a two-way repeated measures ANOVA between sgGFP and the indicated sgRNA conditions. These results were confirmed in at least three independent experiments. Not significant (n.s.).

Fig 4: Intestinal inflammation promotes lipolysis and autophagy in adipose tissues Schematic of experimental design. Sex‐matched and age‐matched wild‐type mice were treated for 5 days with 1.5–2% DSS in drinking water, before switched to water for two more days. Mice were sacrificed on day 7 post‐DSS induction.Body weight development upon DSS treatment (n = 13/group).Tissue weights measured in mesenteric (mWAT) and collective visceral white adipose tissue (visWAT), consisting of gonadal (gWAT), retroperitoneal and omental white adipose tissue on day 7 after start of DSS regime (n = 8/group).Circulating serum levels of FFA during DSS‐induced colitis on day 7 (n = 15/group).Representative immunoblot for key lipolytic enzymes HSL and ATGL protein expression and quantification (n = 5–6/group).Immunoblot analysis of autophagic flux in mWAT (upper panel) and gWAT (lower panel) adipose tissue stimulated ex vivo with lysosomal inhibitors 100 nM Bafilomycin A1 and 20 mM NH4Cl for 4 h or DMSO (Vehicle) (n = 3–4/group).Representative transmission electron microscopy images from mesenteric adipose tissue 7 days post DSS‐induced colitis induction. Lower panel is showing magnification of selected area. White arrows show autophagosomal structures. Atg8 homologs expression was measured by qPCR in visceral adipocytes fraction (left panel) and stromal vascular fraction (right panel) during DSS‐induced colitis (n = 7–8/group).Representative immunoblot for LC3‐I/‐II protein expression and quantification of autophagic flux in gWAT via ex vivo lysosomal inhibition using 100 nM Bafilomycin A1 and 20 mM NH4Cl for 4 h or DMSO (Vehicle). Mice were initially treated with 500 μg anti‐TNFα antibody or isotype control, before administrating DSS in drinking water for 5 days. Mice were sacrificed on day 7 post‐DSS induction (n = 5–6/group).Representative immunoblot for LC3‐I/‐II and ACTIN protein expression and quantification of autophagic flux in creeping fat tissues (CrF) and adjacent mesenteric adipose tissues (Ad. MAT) of Crohn's disease patients (n = 3/group). Additionally, autophagic flux was determined in the mesenteric adipose tissue (MAT) of a colorectal cancer patient as control (dotted line). Data are represented as mean ± s.e.m. (B) Two‐Way repeated measures ANOVA. (C–E, G) Unpaired Student's t‐test. (I) Two‐Way ANOVA. (J) Paired Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure.

Fig 5: Opn3-Dependent Fat Mass Utilization In Vivo and Light- and Opn3-Dependent Lipolysis Activation In Vivo and In Vitro(A) Schematic describing the timeline of a fasting-cold exposure experiment.(B) CBT of fed and fasted Opn3fl/fl and Adipoq-cre; Opn3fl/fl mice during 180 min of cold exposure.(C) Fat mass used by Opn3fl/fl and Adipoq-cre; Opn3fl/fl, fed and fasted, cold-exposed mice.(D) Serum glycerol levels from the same fed and fasted Opn3fl/fl and Adipoq-cre; Opn3fl/fl mice as in (C) after the 180 min of cold exposure.(E) Chart showing that Opn3+/+ mice have elevated cAMP levels in lysates of inWAT during the light phase versus the dark phase, while the cAMP levels in the inWAT of Opn3lacz/lacz mice are similar in both phases, comparable to levels observed in the Opn3+/+ dark phase and significantly lower than those seen in the Opn3+/+ light phase.(F) Cultured in vitro differentiated adipocytes show 475 nm light-dependent, dose-response elevation of cAMP.(G) Two examples of immunoblots showing light-dependent and Opn3-dependent induction of phospho-660-HSL in cultured in vitro differentiated adipocytes. Each set of immunoblots (experiment 1 and experiment 2) was performed using white adipocytes isolated from separate mice.(H) Quantification of phospho-HSL induction in wild-type control (Opn3+/+, n = 5 mice) and Opn3 loss-of-function (Opn3lacz/lacz, n = 3 mice) white adipocytes.(I and J) Quantification of glycerol released from in vitro differentiated Opn3+/+ (I) and Opn3lacz/lacz (J) cells in response to 2 h of blue light stimulation compared with darkness.