CaMKK2 modulates macrophage polarization and is associated with immunometabolic transcriptional changes under high glucose conditions

BACKGROUND Type 2 diabetes mellitus (T2DM) is characterized by chronic metabolic stress and lowgrade inflammation, in which macrophage dysfunction contributes to disease pathogenesis.Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates metabolic and inflammatory processes, but its role in macrophage responses to high glucose (HG) remains unclear.AIM To investigate the effects of CaMKK2 on macrophage polarization and immunometabolic gene expression under HG conditions and assess the involvement of protein kinase B (AKT) signaling.METHODS CAMKK2 overexpression (OE) and knockdown experiments were performed in THP-1derived macrophages under control-and HG conditions.Bulk RNA sequencing analysis, comprising three independent biological samples per group, compared CAMKK2overexpressing cells with untransduced control cells under HG conditions.Polarizationassociated gene expression and protein markers were assessed using quantitative PCR and Western blotting, respectively, and AKT phosphorylation was evaluated by Western blotting.The AKT activator SC79 was used to examine pathway involvement.Additional experiments were conducted in mouse bone marrow-derived macrophages (BMDMs).The in vitro and BMDM experiments included at least three independent biological replicates.Public single-cell RNA-sequencing data from PBMCs of 18 participants (9 patients with T2DM and 9 controls) were additionally analyzed using a donor-level pseudobulk approach to assess monocyte CAMKK2 expression and associated transcriptional pathways. / 42 RESULTSUnder HG conditions, CAMKK2 OE increased CD86 protein abundance (1.49 ± 0.13 vs 1.00 ± 0.05, P = 0.0039) and decreased CD163 protein abundance (0.54 ± 0.14 vs 1.00 ± 0.21, P = 0.0347).Camkk2 deletion attenuated inducible nitric oxide synthase expression in BMDMs from high-fat diet/streptozotocin-treated mice (0.88 ± 0.03 vs 1.16 ± 0.03, adjusted P < 0.0001).Bulk RNA sequencing identified 5043 differentially expressed genes involving inflammatory and metabolic transcriptional programs.CAMKK2 OE reduced the p-AKT/AKT ratio (0.30 ± 0.09 vs 1.00 ± 0.03, P = 0.0002), while SC79 partially attenuated CaMKK2-induced CD86 expression (2.33 ± 0.18 vs 3.18 ± 0.54, adjusted P = 0.0166).Donor-level pseudobulk analysis detected no significant difference in monocyte CAMKK2 expression between patients with T2DM and controls (adjusted P = 0.905). CONCLUSIONCaMKK2 modulates macrophage polarization and is associated with immunometabolic transcriptional changes under HG conditions.Changes in AKT phosphorylation and partial pharmacological rescue suggest that AKT signaling may contribute to these effects.