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 Ca^2+-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: Kinase signaling, Inflammation, Insulin resistance, Glucose homeostasis, Liver steatosis Highlights * • Myeloid-specific CAMKK2 deficiency in mice prevents diet-induced fat accumulation, insulin resistance, and hepatosteatosis. * • CAMKK2 deficiency alters gene expression in macrophages, enhancing fat oxidation and driving an anti-inflammatory phenotype. * • CAMKK2 is an attractive therapeutic target for treating obesity and related metabolic disorders. 1. Introduction Obesity is a major public health concern globally that results from a combination of sustained overnutrition and sedentary behavior, and is associated with the development of life-threatening comorbidities including type 2 diabetes, metabolic-dysfunction associated fatty liver disease (MAFLD), cardiovascular disease, and several cancers [[47][1], [48][2], [49][3]]. A characteristic feature of obesity is the presence of altered immune cell populations and chronic low-grade inflammation in metabolic organs such as white adipose tissue (WAT) and liver, which leads to a systemic pro-inflammatory state that associates with metabolic dysfunction [[50]1,[51]4]. Macrophages are thought to be a major driving force in obesity-induced inflammation [[52]5]. The pathogenesis of obesity is associated with increased accumulation of pro-inflammatory macrophages in WAT and release of inflammatory cytokines and adipokines, which correlate with whole-body insulin resistance. In contrast, lean WAT contains an abundance of anti-inflammatory macrophages that are associated with insulin sensitivity [[53]6]. Consequently, inhibiting pro-inflammatory macrophage infiltration and increasing the predominance of anti-inflammatory macrophages in WAT has been proposed as a therapeutic strategy to treat metabolic disorders caused by obesity-induced inflammation [[54]4]. The cell signaling enzyme, Ca^2+-calmodulin-dependent protein kinase kinase-2 (CAMKK2), is an important regulator of cellular and systemic metabolism that coordinates the function of key metabolic organs including adipose tissue and liver [[55]7,[56]8]. At the cellular level, nutrients and hormones activate CAMKK2 by increasing intracellular Ca^2+ and promoting accumulation of the Ca^2+-calmodulin complex. Germline deletion of Camkk2 protects mice from high-fat diet-induced weight gain, insulin resistance, hepatic steatosis, and hepatocellular carcinoma [[57][9], [58][10], [59][11]]. Although hepatocytes constitute the dominant cell type in the liver, mice with hepatocyte-specific deletion of Camkk2 fail to display the same resistance to hepatic lipid accumulation as their globally deleted counterparts [[60]9,[61]12]. These findings hint at the importance of other cell types in mediating the beneficial effects of Camkk2 deletion on hepatic function and whole-body metabolism. CAMKK2 is highly expressed in macrophages and regulates inflammatory responses but is largely absent in other immune cells of the myeloid lineage [[62]13]. Since pro-inflammatory macrophages recruited to adipose tissue are thought to play a central role in obesity-induced metabolic dysfunction [[63]5], we generated mice with myeloid-specific Camkk2 deficiency to investigate the importance of CAMKK2 function within macrophages for obesity-induced insulin resistance. 2. Results 2.1. Myeloid Camkk2 deficiency protects mice from high-fat diet-induced obesity via an increase in energy expenditure To investigate the role of CAMKK2 signaling in macrophages on whole-body metabolism, we conditionally deleted Camkk2 in myeloid-lineage cells by generating mice homozygous for a floxed allele of Camkk2 and homozygous for a transgene expressing Cre recombinase under the control of the LysM promoter (Camkk2^fl/flLysM-Cre^Tg/Tg termed Camkk2^MKO for simplicity). Camkk2^fl/flLysM-Cre^−/− littermates were used as controls. We confirmed efficient loss of Camkk2 mRNA expression in bone marrow-derived macrophages (BMDMs) from Camkk2^MKO mice and control mice by qPCR ([64]Figure 1A) and immunoblotting ([65]Fig. S1A). Camkk2 deletion had no impact on bone marrow progenitor cell numbers or their ability to differentiate into BMDMs ([66]Fig. S1B–C). Figure 1. [67]Figure 1 [68]Open in a new tab 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. We next studied the effect of myeloid Camkk2 deficiency on whole-body metabolism under conditions of over-nutrition, by monitoring the body weight and adiposity of male Camkk2^MKO mice and age-matched controls fed a high-fat diet (HFD) over a period of 10 weeks. There was no significant difference in body weight prior to commencing the diet. However, Camkk2^MKO mice were resistant to weight gain during HFD feeding ([69]Figure 1B). This effect of weight gain was not due to differences in lean mass ([70]Figure 1C), but rather due to a significant reduction in total fat mass ([71]Figure 1D). Consistent with this observation, Camkk2^MKO also displayed reduced plasma leptin levels, which is an indirect measure of body fat ([72]Figure 1E). The Camkk2^MKO mice also displayed reduced adiponectin levels ([73]Figure 1F). To investigate whether the protection from weight gain and adiposity in Camkk2^MKO mice were associated with changes to energy balance, we measured both food intake and whole-body energy expenditure. Food intake was not different when comparing Camkk2^MKO mice with controls ([74]Figure 1G–H). Indirect calorimetry measurements revealed no differences in respiratory exchange ratio ([75]Figure 1I–J), or total energy expenditure when expressed in absolute terms ([76]Figure 1K–L). However, when we accounted for body weight differences using analysis of covariance (ANCOVA) as recommended by Speakman [[77]14], ANCOVA adjusted energy expenditure was higher in Camkk2^MKO mice compared with control mice ([78]Figure 1M−N). Taken together, these data suggest that Camkk2^MKO mice are resistant to diet-induced obesity due to enhanced energy expenditure rather than reduced food intake. 2.2. Myeloid Camkk2 deficiency improves glycemic control and hepatosteatosis in mice fed a high-fat diet Given the lean phenotype of Camkk2^MKO mice and the well-established link between obesity and poor glycemic control, we next assessed glucose homeostasis in Camkk2^MKO and littermate control mice fed an HFD. Both fasting glucose ([79]Figure 2A) and insulin ([80]Figure 2B) were lower in Camkk2^MKO mice compared with control mice. We next challenged the mice by measuring glucose and insulin tolerance. Camkk2^MKO displayed a marked improvement in both glucose ([81]Figure 2C) and insulin ([82]Figure 2D) tolerance compared with controls. To further investigate the improvements in glucose clearance and insulin responsiveness in Camkk2^MKO mice, we performed hyperinsulinemic-euglycemic clamp experiments, incorporating tritium (^3H) and carbon-14 (^14C) radioactive tracers to determine tissue-specific insulin sensitivities. Camkk2^MKO mice displayed a clear (p < 0.001) increase in glucose infusion rate (GIR) compared with control ([83]Figure 2E). Furthermore, glucose disposal rate (GDR) was increased ([84]Figure 2F) as was glucose uptake (p < 0.01) by epididymal ([85]Figure 2G) WAT. While not statistically significant, hepatic glucose production (HGP) tended to be reduced ([86]Figure 2H), while skeletal muscle (gastrocnemius) glucose uptake trended upwards ([87]Figure 2I) in Camkk2^MKO mice compared with controls. Plasma triglyceride levels were also increased in Camkk2^MKO mice ([88]Figure 2J), suggesting increased lipolysis. These findings demonstrate that mice lacking CAMKK2 expression in macrophages are protected from impairments in glucose homeostasis induced by a high-fat diet and that the liver, WAT and skeletal muscle undergo wholesale adaptations that improve energy balance. Figure 2. [89]Figure 2 [90]Open in a new tab 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. Given the marked decrease in fat mass and trend towards decreased HGP in the Camkk2^MKO mice compared with control, we next examined whether these mice were also protected from high-fat diet induced hepatosteatosis. Following HFD feeding, livers were harvested, paraffin embedded, sectioned and analyzed for hematoxylin and eosin (H&E) and Oil Red O staining (ORO) ([91]Figure 3A). Macro vesicular steatosis ([92]Figure 3B), ORO-stained area ([93]Figure 3C), and liver triglycerides ([94]Figure 3D) were all significantly reduced (p < 0.05) in Camkk2^MKO mice compared with control mice, suggesting that myeloid-specific loss of Camkk2 protects against diet-induced lipid accumulation in the liver. Figure 3. [95]Figure 3 [96]Open in a new tab 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. 2.3. Myeloid-deficient Camkk2 mice fed a HFD have reduced pro-inflammatory CD11c macrophages in eWAT and an anti-inflammatory transcriptional profile Epididymal white adipose tissue (eWAT) secretes an array of cytokines to regulate the metabolism of organs such as liver and skeletal muscle in HFD-induced obesity [[97]15]. Accordingly, we next examined macrophage subtypes in eWAT in our mice by flow cytometry ([98]Fig. S2A). Although consumption of a HFD failed to significantly alter the population of F4/80^+ CD11b^+ macrophages when comparing Camkk2^MKO mice with control mice ([99]Figure 4A), myeloid-specific loss of Camkk2 markedly decreased (p < 0.01) the percentage of CD11c^+/CD206^- macrophages ([100]Figure 4B), while conversely increased (p < 0.01) the percentage of CD206^+/CD11c^− macrophages in eWAT tissue ([101]Figure 4C). We next examined an array of chemokines and adipokines within eWAT. Monocyte chemoattractant protein-1 (MCP-1), a key chemokine that regulates migration and infiltration of macrophages into tissues, was markedly reduced (p < 0.01) in eWAT from Camkk2^MKO mice ([102]Figure 4D). Adiponectin, Angiopoietin-like protein 3 (ANGPT-L3), Fibroblast growth factor-1 (FGF-1), Leptin and Serpine-1 were also reduced (p < 0.05), while Insulin-Like Growth Factor Binding Proteins-2 and -6 (IGFBP-2, IGFBP-6) were increased (p < 0.01) in Camkk2^MKO mice ([103]Figure 4D). Figure 4. [104]Figure 4 [105]Open in a new tab 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. Given the shift towards increased levels of CD206 macrophages and downregulation of MCP-1 expression in eWAT, we next examined whether Camkk2 deficient macrophages more broadly exhibited anti-inflammatory gene transcriptional profiles. Accordingly, we obtained BMDMs from Camkk2^MKO and control mice, treated them with lipopolysaccharide (LPS) and interferon-γ (IFNγ) and performed RNAseq experiments. LPS/IFNγ treatment induced a rapid increase in Camkk2 expression in BMDMs from control mice, but not from Camkk2^MKO mice ([106]Fig. S2B). Principal component analysis ([107]Figure 4E), and fold changes in gene expression via heatmap ([108]Figure 4F) and volcano ([109]Figure 4G) plots are shown. Pathway enrichment analyses revealed that processes associated with inflammation and oncogenesis such as inflammatory response, TNF signaling, interferon gamma response, and KRAS signaling, were all downregulated in Camkk2^MKO relative to control mice, while metabolic pathways associated with fatty acid metabolism, xenobiotic metabolism and bile acid metabolism were all upregulated in Camkk2^MKO mice relative to controls ([110]Figure 4H). Using the STRING database to collect, score and integrate protein–protein interactions, a cluster of key inflammatory response genes were found to be uniformly down-regulated in BMDMs obtained from Camkk2^MKO treated with LPS and IFNγ, relative to control BMDMs ([111]Figure 4I). These data demonstrate that loss of CAMKK2 from BMDMs reprograms these cells to an inflammatory resistant state. 2.4. Myeloid-deficient Camkk2 mice fed a HFD display a beiging transcriptional profile in both visceral and subcutaneous WAT Given the data we had generated to this point, we next performed RNAseq experiments on eWAT samples obtained from Camkk2^MKO and control mice fed an HFD. Principal component analysis ([112]Figure 5A), and fold changes in gene expression via heatmap ([113]Figure 5B) and volcano ([114]Figure 5C) plots are shown. Importantly, pathway enrichment analyses revealed that processes associated with fibrosis and inflammation such as epithelial to mesenchymal transition, hypoxia and TNF signaling were all downregulated in Camkk2^MKO mice relative to control mice, while pathways associated with increased energy utilization such as fatty acid metabolism, oxidative phosphorylation and glycolysis were all upregulated in Camkk2^MKO mice ([115]Figure 5D). STRING analysis indicated protein–protein interactions of key factors associated with fatty acid metabolism and beiging of WAT such as Uncoupling Protein-1 (UCP1), Cell Death Activator-A (CIDEA) and Very Long Chain Fatty Acid Elongase-3 (ELOVL3) enriched in Camkk2^MKO eWAT ([116]Figure 5E). We also performed RNAseq analysis on subcutaneous inguinal WAT (iWAT) from Camkk2^MKO and control mice fed a HFD, and observed a similar upregulation of proteins involved fatty acid metabolism and beiging relative to control mice ([117]Figure 5F–J). Taken together, these data indicate that myeloid-specific loss of CAMKK2 expression decreases adiposity in HFD-fed mice by transcriptionally regulating pathways that increase fatty acid oxidation in both visceral and subcutaneous WAT. Figure 5. [118]Figure 5 [119]Open in a new tab 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. 2.5. Macrophages lacking Camkk2 display increased rates of fatty acid oxidation Given the transcriptional profile of BMDMs and eWAT obtained from Camkk2^MKO relative to control mice described above, we next performed functional analyses in these macrophages. Naïve BMDMs obtained from Camkk2^MKO mice displayed higher (p < 0.01) palmitate oxidation relative to control BMDMs ([120]Figure 6A). We next performed Seahorse assays to determine the oxygen consumption rates (OCR) of the BMDMs. When treated with 200 μM palmitate, both maximal respiration and spare capacity were increased (p < 0.01) in Camkk2^MKO relative to control BMDMs ([121]Figure 6B–C). These data indicated a switch in the fuel preferences and ability of BMDMs from Camkk2^MKO mice to process select