Macrophage CREBZF Orchestrates Inflammatory Response to Potentiate Insulin Resistance and Type 2 Diabetes

Abstract Chronic adipose tissue inflammation accompanied by macrophage accumulation and activation is implicated in the pathogenesis of insulin resistance and type 2 diabetes in humans. The transcriptional coregulator CREBZF is a key factor in hepatic metabolism, yet its role in modulating adipose tissue inflammation and type 2 diabetes remains elusive. The present study demonstrates that overnutrition‐induced CREBZF links adipose tissue macrophage (ATM) proinflammatory activation to insulin resistance. CREBZF deficiency in macrophages, not in neutrophils, attenuates macrophage infiltration in adipose, proinflammatory activation, and hyperglycemia in diet‐induced insulin‐resistant mice. The coculture assays show that macrophage CREBZF deficiency improves insulin sensitivity in primary adipocytes and adipose tissue. Mechanistically, CREBZF competitively inhibits the binding of IκBα to p65, resulting in enhanced NF‐κB activity. In addition, bromocriptine is identified as a small molecule inhibitor of CREBZF in macrophages, which suppresses the proinflammatory phenotype and improves metabolic dysfunction. Furthermore, CREBZF is highly expressed in ATM of obese humans and mice, which is positively correlated with proinflammatory genes and insulin resistance in humans. This study identifies a previously unknown role of CREBZF coupling ATM activation to systemic insulin resistance and type 2 diabetes.

Isolation and differentiation of primary white adipocytes.Subcutaneous fat isolated from 4-to 6week-old mice was digested for 40-60 min at 37°C with 1 mg/ml collagenase solution.After filtrated and centrifuged, the SVF cells were plated in DMEM supplemented with 10% FBS and 1% P/S.When SVF reached confluence, the cells were passaged.When cells reached confluence again, replaced the medium to induction medium (1 μg/μl insulin, 0.5 mM IBMX, 1 μM dexamethasone and 0.5 μM rosiglitazone) and cultured the cells for 48 h.Then changed to a maintenance medium (1 μg/μl insulin and 1 μM rosiglitazone) for another 7-8 days to obtain mature white adipocytes.
Coculture of primary white adipocytes with BMDMs conditioned media.The protocol is modified from the previously description 2 .BMDMs were treated with 100 ng/ml LPS for 6 h.Then the medium of BMDMs was changed to a serum-free medium.After incubated for another 12 h, the medium was harvested and mixed with fresh serum-free medium at the ratio of 1:1 as BMDMs conditioned media.
The mature adipocytes were pretreated with serum-free medium or BMDMs conditioned media for 12 h followed by 100 nM insulin treatment for 15 min or 30 min.

Coculture of white adipose tissues with BMDMs.
The protocol is modified from the previously description 3 .Briefly, isolated 0.2 g intact epididymal white adipose tissue from wild-type mice and rinsed it using PBS.Put the tissue into a well in which bone marrow-derived macrophages were precultured.Then treated the cocultured system with 100 ng/ml LPS.After 24 hours, the cocultured system was stimulated by 100 nM insulin for 15 min or 30 min.

Metabolic phenotyping.
For glucose tolerance tests (GTT), mice were fasted for 16 h followed by intraperitoneal injection with glucose solution (1 g/kg body weight).For insulin tolerance tests (ITT), mice were fasted for 6 h, and intraperitoneally injected with 1 U/kg insulin solution.Blood glucose was determined at 0, 15, 30, 60, 90, and 120 min after injection of glucose or insulin 6 .Triglycerides determination kit (Thermo Fisher Scientific), cholesterol determination kit (Thermo Fisher Scientific), FFA assay kit (Solarbio), and insulin ELISA kit (Millipore) were used for plasma, respectively according to the manufacturer's instructions.

Macrophage chemotaxis assay.
BMDMs from WT and CREBZF MKO mice were seeded into the upper wells of a transwell chamber plate (Corning).The macrophages were challenged with 20 ng/ml CCL2 or fresh adipose tissue lysates from obese mice in the lower wells.After incubated for 8 hours, the membrane inserts were fixed and stained by crystal violet.The transmigrated cells on the lower surface of the membrane were imaged by a light microscope and counted by ImageJ software.
ChIP sequencing and bioinformatics analysis.The preparation for ChIP sequencing samples was previously described 7 .BMDMs from CREBZF MKO mice were isolated and infected with adenovirus expressing GFP or myc-tagged CREBZF and followed treatment for 6 hours with LPS.The chromatin was immunoprecipitated by anti-body against myc (Cell Signaling Technology).DNA was then purified by QIAquick columns (QIAGEN) and quantified with Qubit Fluorometer (Thermo Fisher Scientific).ChIP DNA Libraries were prepared by VAHTS® Universal DNA Library Prep Kit for Illumina V3 (Vazyme) according to the manufacturer's protocols.The sequence reads generated by Illumina sequencing were mapped to the mouse genome (mm10) using BWA algorithm, followed by peak calling with MACS2.
The differential peaks in promoter regions were identified over the input control, then were analyzed for determining TF motifs with JASPAR database.And the differential peaks-related genes were analyzed for KEGG pathway analysis by DAVID.
RNA sequencing and bioinformatics analysis.RNA sequencing and data analysis were performed by Majorbio (Shanghai, China).Total RNA was extracted by using TRIzol Reagent (Life Technologies), and the sequencing library was constructed.The expression level of each transcript was calculated according to the transcripts per million reads (TPM) method, then DEGs (differential expression genes) were identified and plotted heatmap.The gene sets were performed GO analysis, KEGG analysis 8 , and so forth.Stably transfected CREBZF-promoter-luciferase cell line.To establish stably expressing CREBZFpromoter-luciferase cells, pGL3-CREBZF-promoter and pcDNA3.1 were co-transfected to Huh7.After 24 h, G418 at 800 μg/ml was added for 7 days to select stably transfected cells.Monoclonal cell lines were isolated and further identified by luciferase assay.
High-throughput screening.FDA approved drug library containing 640 compounds (Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China) was diluted in DMSO.Stably transfected CREBZF-promoter luciferase Huh7 cells were seeded in 384-well plates by Multidrop Combi (Thermo Fisher Scientific) and treated with 10 μM of each indicated compound.DMSO treatment was used as a control.After 48 hours, the activities of CREBZF promoter were measured by luciferin reactions (Promega) and cell viabilities were measured by Presto Blue (Thermo Fisher cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) in a humidified 5% CO2 incubator at 37°C as previously described 10 .
Protein-protein docking.The crystal structure of the IκBα/NF-κB complex 14 was visualized by PyMOL software.The structure of CREBZF was predicted by AlphaFold.The crystal structure of the p65/p50 heterodimer bound to the DNA complex was studied 15 .CREBZF and the p65/p50 heterodimer were docked by ClusPro2.0,and then the structure was visualized by PyMOL software.

Fig. S1 .
Fig.S1.Myeloid CREBZF deficiency dose not impair immune system development.(A) The percentages of macrophages, neutrophils, and dendritic cells in the bone marrow.(B) The percentages

Fig. S10 .
Fig.S10.CREBZF is correlated with inflammation and metabolic alterations in human adipose

Fig. S11 .
Fig.S11.The proposed model for macrophage CREBZF in regulating inflammation and insulin Dual luciferase assays.Cells were co-transfected with plasmids encoding p65 or IκBα as indicated, luciferase reporter plasmids containing different promoter sequences, and Renilla luciferase plasmids as control.4xNFκB Luc plasmid was purchased from ADDGENE.Renilla luciferase plasmid and pGL3basic plasmid were purchased (Promega).After 36-48 hours, the activities of Firefly and Renilla luciferase in the lysates were measured with Dual luciferase assays (Promega).The luminescence was measured using an Infinite 2000pro plate reader (TECAN).The results were normalized to Renilla luciferase activity.