Activation of IRF1 in Human Adipocytes Leads to Phenotypes Associated with Metabolic Disease

Figure 1. Primary and ASC-Adipocyte Integrity and Transcriptional Profiling (A) Primary adipocytes isolated from whole adipose. Adipocytes were stained with DAPI to visualize the DNA (blue), BoDIPY to stain the lipid droplet (green), and LavaCell to stain the plasma membrane (red), and visualized by wide-field microscopy. (B) ASCs were differentiated into adipocytes and stained with DAPI to visualize the DNA (blue), BoDIPY to stain lipids (green), and an antibody to visualize C/EBPα (red). (C) Hierarchical clustering and heatmap representation of transcriptional profiles (mRNA) from ASCs, ASC adipocytes, and primary adipocytes. Probe sets are colored according to expression level. (D) Gene expression of IRF1 in ASCs (n = 3), ASC adipocytes (n = 3), and primary adipocytes (n = 4). (E) Gene expression of IRF1 in isolated subcutaneous adipocytes of lean (n = 10) and obese (n = 10) individuals. For all graphs, error bars represent SD, experiments were performed in biological triplicates and statistically significant p values are denoted by asterisks (∗p ≤ 0.05, ∗∗p ≤ 0.005).

Figure 2. IRF1 Expression Leads to an Inflammatory Gene Signature (A) Experimental scheme to ectopically express IRF1 in mature ASC adipocytes. (B) ChIP-qPCR (n = 3) was performed for IRF1 at putative binding sites (H3K4me1 bound IRF1 motif) (PARP14, IFIT3, ISG15). OAS3 and ZNF74 represent a negative control site where IRF1 is not predicted to bind. Signals are expressed as percentage of total chromatin input. The heatmap represents expression levels of these genes in IRF1 and rtTA adipocytes. (C) Hierarchical clustering and heatmap representation of transcriptional profiles (mRNA) from rtTA adipocytes, IRF1 adipocytes, and primary adipocytes. Probe sets are colored according to expression level. The highest expressed 30% of genes in IRF-1 adipocytes have been expanded to highlight their inflammatory nature. Error bars represent SD, experiments were performed in biological triplicates, and statistically significant p values are denoted by asterisks (∗p ≤ 0.05, ∗∗p ≤ 0.005).

Figure 3. IRF1 Expression Impairs Adipocyte Metabolism and Affects Lipid Composition (A) Multiplex ELISA assay was utilized to measure cytokines secreted into medium by IRF1 adipocytes compared with control adipocytes. (B) Glycerol release into the medium in response to forskolin (FSK)-activated lipolysis was measured in IRF1 and rtTA adipocytes. (C) Radiolabeled glucose uptake was measured in response to insulin. (D) After 5 days of IRF1 expression, IRF1 adipocytes become unilocular. Lipid droplet size was measured after staining with the lipid dye BoDIPY (green) and the nuclear dye DAPI (blue). CPM, counts per minute. (E) Total intracellular triglycerides (TG) were measured in IRF1 and rtTA adipocytes. (F) Tandem mass spectrometric profiles of triglyceride composition for both IRF1 and rtTA adipocytes. Error bars represent SD, experiments were performed in biological triplicates, and statistically significant p values are denoted by asterisks (∗p ≤ 0.05, ∗∗p ≤ 0.005). n = 3 for each experiment.

Figure 4. IRF1 Leads to Increased Inflammation In Vivo (A) 3T3-F442A cells were transduced with either rtTA alone, or human IRF1 (hIRF1) containing lentiviral particles, and were injected into 6-week-old nude mice (n = 4). Six weeks after injection, ectopic subcutaneous fat pads (dashed circles) were excised. (B) Ectopic fat explants (EXP) were stained with DAPI (blue) and probed with α-F4/80 (green) and α-PLIN1 (red). (C) F4/80-positive cells were counted relative to DAPI staining foci in hIRF1 explants versus rtTA explants and autologous WAT (see Figure S3C). A minimum of 104 DAPI foci were counted for each experiment. (D) qPCR was used to assess expression of hIRF1 and inflammation-associated mouse genes in both explants and autologous WAT (n = 3). Error bars represent SD, experiments were performed in biological triplicates, and statistically significant p values are denoted by asterisks (∗p ≤ 0.05, ∗∗p ≤ 0.005).