Myeloid-specific CAMKK2 deficiency protects against diet-induced obesity and insulin resistance by rewiring metabolic gene expression and enhancing energy expenditure

Abstract

Objective: Obesity is associated with chronic, low-grade inflammation in metabolic tissues such as liver, adipose tissue and skeletal muscle implicating insulin resistance and type 2 diabetes as inflammatory diseases. This inflammatory response involves the accumulation of pro-inflammatory macrophages in these metabolically relevant organs. The Ca²⁺-calmodulin-dependent protein kinase kinase-2 (CAMKK2) is a key regulator of cellular and systemic energy metabolism, and a coordinator of macrophage-mediated inflammatory responses. However, its role in obesity-associated metabolic dysfunction is not fully defined. The aim of this study was to determine the contribution of CAMKK2 to the regulation of inflammation and systemic metabolism during diet-induced obesity.

Methods: Mice with myeloid-specific deletion of Camkk2 were generated and challenged with a high-fat diet. Metabolic phenotyping, histological analyses, and transcriptomic profiling were used to assess whole-body metabolism, liver lipid accumulation, and gene expression in macrophages and adipose tissue.

Results: Myeloid-specific Camkk2 deficiency protected mice from high fat diet-induced obesity, insulin resistance and liver steatosis. These protective effects were associated with rewiring of metabolic and inflammatory gene expression in both macrophages and adipose tissue, along with enhanced whole-body energy expenditure.

Conclusions: Our data establish CAMKK2 as an important regulator of macrophage function and putative therapeutic target for treating obesity and related metabolic disorders.

Keywords: Glucose homeostasis; Inflammation; Insulin resistance; Kinase signaling; Liver steatosis.

Figure 1

Camkk2^MKO mice are protected from diet-induced obesity. qPCR analysis of Camkk2 mRNA expression in bone marrow-derived macrophages (A), weekly body mass progression (B), lean mass (C), fat mass (D), plasma leptin (E), and plasma adiponectin levels (F) of Camkk2^MKO and control mice after 12 weeks on HFD. Cumulative food intake over a 72-hour period (G), average food intake within the light and dark cycle (H), respiratory exchange ratio over a 72-hour period (I), average respiratory exchange ratio within the light and dark cycle (J), energy expenditure over a 72-hour period (K), average energy expenditure within the light and dark cycle (L), regression plot comparing average daily energy expenditure measured over 72 h to body weight (M), and ANCOVA-adjusted energy expenditure (N), of Camkk2^MKO and control mice measured in metabolic cages over a 72 h period after 12 weeks on an HFD diet. For (A), (C), (D), (E), and (F): Unpaired t-test was used to analyze the data. For (B): Two-way repeated measures ANOVA was performed followed by Fisher's LSD post-hoc test; Control, n = 9, Camkk2^MKO, n = 10. For (H), (J), (L) and (N): Two-way ANOVA was used followed by Sidak's post-hoc multiple comparisons test. Data are presented as means ± SEM. ∗P < 0.05, ∗∗∗∗P < 0.0001.

Figure 2

Camkk2^MKO mice display improved glycemic control on HFD. Fasting blood glucose (A), plasma insulin (B), glucose tolerance test (C), insulin tolerance test (D), and hyperinsulinemic-euglycemic clamp data showing glucose infusion rate (E), glucose disposal rate (F), eWAT glucose uptake (G), hepatic glucose production post-clamp (H) and skeletal muscle glucose uptake (I), plasma triglyceride levels (J), in Camkk2^MKO and control mice after 12 weeks on HFD. For (A), (B), (E), (F), (G), (H), (I) and (J): Unpaired t-test was used to analyze the data. For (C) and (D): Two-way repeated measures ANOVA was performed followed by Fisher's LSD post-hoc test; Control, n = 10, Camkk2^MKO, n = 9. Data are presented as means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 3

Camkk2^MKO mice are protected from HFD-induced hepatic steatosis. Representative images of liver sections stained with H&E and Oil Red O (A), liver Oil Red O quantification (B), macro vesicular steatosis score (C), and liver triglyceride content (D), of Camkk2^MKO and control mice after 12 weeks on HFD. Scale bars for (A): 100 μM. For (B), (C), and (D): Unpaired t-test was used to analyze the data. Data are presented as means ± SEM. ∗P < 0.05.

Figure 4

Camkk2^MKO mice on HFD have reduced CD11c⁺ inflammatory macrophages and an anti-inflammatory transcriptional profile. Quantification of F4/80⁺CD11b⁺ macrophages as a percentage of viable CD45⁺ cells (A), CD11c⁺/CD206^- inflammatory macrophages as a percentage of F4/80⁺CD11b⁺ macrophages (B), CD206⁺/CD11c⁻ anti-inflammatory macrophages as a percentage of F4/80⁺CD11b⁺ macrophages (C), and adipokine expression (D) in eWAT extracted from Camkk2^MKO and control mice after 12 weeks on HFD. RNAseq data showing principal component analysis (E), heatmap and volcano plots of changes in gene expression (F and G), pathway enrichment analysis (H), and STRING protein–protein interaction analysis (I) of differentially expressed genes in naïve BMDMs treated with lipopolysaccharide (LPS) and interferon-γ (IFNγ) from Camkk2^MKO and control mice. For (A), (B), and (C): Unpaired t-test was used to analyze the data. For (D), two-way ANOVA was used followed by Sidak's post-hoc multiple comparisons test. Data are presented as means ± SEM. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 5

Camkk2^MKO mice fed a HFD display a beiging transcriptional profile in white adipose tissue. RNAseq data showing principal component analysis (A), heatmap and volcano plots of changes in gene expression (B and C), pathway enrichment analysis (D), and STRING protein–protein interaction analysis (E) of differentially expressed genes in eWAT from Camkk2^MKO and control mice after 12 weeks on HFD. RNAseq data showing principal component analysis (F), heatmap and volcano plots of changes in gene expression (G and H), pathway enrichment analysis (I), and STRING protein–protein interaction analysis (J) of differentially expressed genes in iWAT from Camkk2^MKO and control mice after 12 weeks on HFD.

Figure 6

Macrophages lacking CAMKK2 display increased rates of fatty acid oxidation. Palmitate oxidation (A), oxygen consumption rate (B), basal respiration, ATP-linked respiration, maximal respiration, and spare capacity (C), schematic explaining metabolic tracing with ¹³C palmitate (D), labeling pattern of ¹³C palmitate into TCA metabolites (E and F), citrate synthase assay (G), mitochondrial staining with Mitotracker Red CMXRos (H and I), and qPCR analysis of Ppargc1a gene expression (J) in naïve BMDMs from Camkk2^MKO and control mice. Scale bars for (H): 10 μM. For (A), (G), (I) and (J): Unpaired t-test was used to analyze the data. For (C) and (E), two-way ANOVA was used followed by Sidak's post-hoc multiple comparisons test. Data are presented as means ± SEM. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.0001.