Macrophage-Specific Progranulin Deficiency Prevents Diet-Induced Obesity through the Inhibition of Hypothalamic and Adipose Tissue Inflammation
Article information
Abstract
Background
Chronic low-grade inflammation in multiple metabolic organs contributes to the development of insulin resistance induced by obesity. Progranulin (PGRN) is an evolutionarily-conserved secretory protein implicated in immune modulation. The generalized deletion of the PGRN-encoded Grn gene improves insulin resistance and glucose intolerance in obese mice fed a high-fat diet (HFD). However, it remains unclear which cells or organs are responsible for the beneficial metabolic effect of Grn depletion.
Methods
Considering the critical role of macrophages in HFD-induced obesity and inflammation, we generated mice with a macrophage-specific Grn depletion (Grn-MΦKO mice) by mating lysozyme M (LysM)-Cre and Grn-floxed mice. Body weight, food intake, energy expenditure, and glucose and insulin tolerance were compared between Grn-MΦKO mice and their wild-type(WT) controls under normal chow diet (NCD)- or HFD-fed conditions. We also examined macrophage activation and inflammation-related gene expression in the visceral adipose tissue and hypothalamus along with insulin and leptin signaling.
Results
Grn-MΦKO mice showed no alteration in metabolic phenotypes under NCD-fed conditions. However, upon HFD feeding, these mice exhibited less weight gain and improved glucose and insulin tolerance compared to WT mice. Moreover, HFD-induced macrophage activation and proinflammatory cytokine expression were significantly reduced in both the adipose tissue and hypothalamus of Grn-MΦKO mice, while HFD-induced impairments in leptin and insulin signaling showed improvement.
Conclusion
Macrophage-derived PGRN and possibly other Grn products play a critical role in the development of HFD-induced obesity, tissue inflammation, and impaired hormonal signaling in both central and peripheral metabolic organs.
Highlights
• The absence of macrophage Grn has no impact on metabolism under a normal diet.
• Macrophage Grn deficiency prevents high-fat diet-induced obesity.
• High-fat diet-induced changes in adipose tissue are mediated by macrophage Grn.
• Hypothalamic inflammation in obese mice is improved by macrophage Grn depletion.
INTRODUCTION
Obesity is a multifaceted disease influenced by genetic, environmental, and behavioral factors. It leads to numerous health issues, including type 2 diabetes mellitus. The cumulative evidence to date suggests that excessive fat accumulation during the course of obesity progression induces low-grade chronic inflammation across various organs [1]. This metabolic inflammation, termed ‘metainflammation,’ is fundamental to the metabolic complications associated with obesity [1]. For instance, adipose tissue fat overload triggers inflammation within adipose tissue [2]. This process leads to insulin resistance and, ultimately, the onset of type 2 diabetes mellitus [2,3]. Adipose tissue macrophages (ATMs) play a crucial role in the development of obesity-associated adipose tissue inflammation by sensing adipocyte-released fatty acids and by releasing proinflammatory cytokines [4,5].
Similar to adipose tissue, an inflammatory response to fatty acids readily occurs in the hypothalamus, a crucial organ in regulating systemic energy and glucose homeostasis. Interestingly, hypothalamic metainflammation is most pronounced in the hypothalamic arcuate nucleus (ARC) [6], which is primarily responsible for sensing the metabolic state of peripheral organs and modulating feeding behaviors and energy metabolism [7]. The microglia, the brain’s resident myeloid cells, play a pivotal role in the hypothalamic inflammation observed in obesity [6,8]. Additionally, hypothalamic perivascular macrophages, another type of myeloid cell with a different cellular origin, become activated and expand their population upon chronic exposure to a high-fat diet (HFD), exhibiting a delayed time course compared to that of microglia [9]. During the progression of obesity, these macrophages sustain hypothalamic inflammation through the release of proinflammatory cytokines and nitric oxide, as well as increased vascular permeability and lipid flux into the hypothalamus [9]. This process boosts the activation of microglia and astrocytes, further exacerbating inflammation. Hypothalamic inflammation has a causative role in obesity and its metabolic complications by disrupting the ability of hypothalamic neurons to perceive metabolic hormonal signals such as leptin [10].
Progranulin (PGRN) is an evolutionally conserved secretory protein encoded by the granulin gene (Grn). PGRN is a precursor protein consisting of 7.5 granulin (GRN) (A, B, C, D, E, F, G, and p) motifs and is proteolytically processed to give rise to GRNs with different biological properties [11]. PGRN has drawn attention in the neuroscience research field as its insufficiency is closely related to a number of neurodegenerative disorders [12]. Moreover, PGRN is known to play crucial roles in immune modulation in various inflammatory contexts [13]. PGRN has anti-inflammatory effects, while GRNs promote inflammation [14-16]. Considering their contrasting actions, the balance between PGRN and GRNs may be important for regulating inflammation and immune responses.
Interestingly, PGRN has been identified as an adipokine, with an expression profile that is enhanced by dexamethasone and tumor necrosis factor-α (TNFα), and repressed by the insulin-sensitizing agent pioglitazone [17]. Global Grn knockout (Grn−/−) mice show an attenuation of HFD-induced insulin resistance, adipocyte hypertrophy, ATM accumulation, and obesity [17]. Conversely, a 14 day-administration of recombinant PGRN was found to cause insulin resistance in normal chow diet (NCD)-fed wild-type (WT) mice [17]. Mechanistically, PGRN inhibits insulin signaling by increasing interleukin-6 (IL-6) production in adipose tissues. PGRN was shown to be expressed in both adipocytes and macrophages, both of which are increased under HFD conditions [17]. In addition, a recent single-nucleus RNA sequencing data set from mouse adipose tissues has revealed that PGRN is also expressed in other types of adipose tissue cells such as adipose stem and progenitor cells, immune cells (T or B lymphocytes, dendritic cells, and natural killer [NK] cells), vascular endothelial and smooth muscle cells, and female epithelial cells [18]. However, it remains unclear which cell type-derived PGRN plays a critical role in insulin resistance and obesity development.
Additionally, PGRN is known to be expressed in neurons and glial cells, including ionized calcium-binding adaptor molecule 1 (Iba1)+ myeloid cells and tanycytes, in the hypothalamus, a key center for energy homeostasis [19]. Single-cell sequencing data from the mouse ARC has revealed that PGRN is expressed in all types of hypothalamic cells, with the highest expression found in microglia, the principal central nervous system immune cells [20].
Given the potential contribution of macrophages and their secretory biological molecules to obesity and its metabolic complications, our study aimed to further elucidate the role of macrophage-derived PGRN in the homeostatic regulation of energy and glucose metabolism and diet-induced obesity (DIO). Our findings indicate that macrophage Grn deficiency replicates the metabolic phenotypes observed in Grn−/− mice, accompanied by significant improvements in inflammation and hormonal signaling in both adipose tissue and the hypothalamus.
METHODS
Animals
Lysozyme M (LysM)-Cre mice (The Jackson Laboratory, Bar Harbor, ME, USA, #004781) in a C57BL/6 genetic background were crossed with Grnf/f (The Jackson Laboratory, #013174) mice to produce macrophage-specific Grn depletion (Grn-MΦKO) mice. Grnf/f littermates were used as Grn-WT mice. For macrophage labeling, LysM-Cre mice or Grn-MΦKO mice were bred with mice carrying a tandem dimer tomato (tdTomato) or enhanced green fluorescence protein (eGFP) reporter allele with an upstream loxP-flanked STOP codon (The Jackson Laboratory, #007914 or #007676). The animals were housed under controlled temperature conditions (22°C±1°C) and 12-hour light-dark cycle (lights on at 8:00 AM). The mice had ad libitum access to a standard chow diet (containing 12.5% of calories from fat; Cargill Agri Purina, Seongnam, Korea) and water, unless otherwise specified. To induce DIO, mice were fed a HFD containing 58% fat (Research Diets, New Brunswick, NJ, USA) starting at 7 weeks of age.
Confirmation of macrophage Grn knockout
To confirm the Grn gene knockout in macrophages, blood samples (200 μL) were collected from the retro-orbital plexus of the mice (LysM-Cre;tdTomato or Grn-MΦKO;tdTomato) and then incubated with 1 mL of 1X red blood cell lysis buffer (Invitrogen, Waltham, MA, USA, #00-4333) for 10 minutes. After washing with 1X phosphate buffered saline (PBS) three times, the cells were centrifuged (300 ×g) at 4°C for 5 minutes and resuspended in 2% fetal bovine serum-containing PBS. The cells were then incubated with PGRN antibody (1:200, R&D Systems, Minneapolis, MN, USA, #AF2557) for 30 minutes followed by fluorescein Isothiocyanate (FITC)-conjugated anti-sheep secondary antibody (1:1,000, Thermo Fisher, Waltham, MA, USA, #A16042) for 20 minutes. The cells were subsequently subjected to fluorescence-activated cell sorting (FACS) using a BD FACS Aria II Cell Sorter (BD Biosciences, Franklin Lakes, NJ, USA), where they were sorted based on tdTomato and FITC fluorescence. For tdTomato fluorescence compensation, non-fluorescent blood samples were obtained from LysM-Cre littermates (Supplementary Fig. 1A). Successful Grn depletion in macrophages was confirmed by a significant depletion of PGRN+ blood myeloid cells (Supplementary Fig. 1). To ensure successful Grn depletion in blood monocytes, blood was collected from Grn-WT mice and Grn-MΦKO mice and processed as described above. The cells were then incubated with CD11b-allophycocyanin-cyanine 7 (APC-Cy7) (1:100, rat, BD Biosciences, #557657) and lymphocyte antigen 6 family member C (Ly6C)-phycoerythrin (1:100, rat, Thermo Fisher, #12-5932-80) antibodies for 30 minutes. Blood monocytes were then gated and sorted via FACS. The successful depletion of Grn in CD11b+ Ly6C+ monocytes was confirmed by Grn quantitative polymerase chain reaction (qPCR) analysis.
Metabolic phenotyping
Food intake levels and body weights were monitored weekly after weaning until the indicated ages. Body compositions (lean mass and fat mass) were measured using dual X-ray absorptiometry (iNSiGHT VET DXA, OsteoSys, Seoul, Korea) on the day of energy expenditure (EE) measurement. EE and locomotor activity were determined using a comprehensive lab monitoring system (CLAMS) (Columbus Instruments, Columbus, OH, USA). Mice were placed in the CLAMS chambers for 48 hours to adapt to the conditions before measurement. During this period, day-night cycles were consistent with those during the initial housing of the animals, and food was provided as pellets on the floor of the CLAMS cages. All EE data were normalized to the lean mass values.
Glucose and insulin tolerance tests
For the glucose tolerance test (GTT), D-glucose (1 g/kg, Sigma, St. Louis, MO, USA) was orally administered to overnight-fasted mice. For the insulin tolerance test (ITT), insulin (Humulin-R 0.25 U/kg, Eli Lilly, Indianapolis, IN, USA) was injected into the peritoneum of the overnight-fasted mice. Blood samples were obtained from tail veins for glucose measurements at 0, 15, 30, 60, and 120 minutes (for GTT) or 0, 30, 60, 90, and 120 minutes (for ITT) after injections. Blood glucose levels were measured using a glucometer (ACCU-CHEK, Aviva Plus System, Indianapolis, IN, USA).
Flow cytometry analysis of adipose tissue macrophages
Epididymal adipose tissues were dissected, chopped, and then incubated in collagenase buffer (0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], 0.125 M NaCl, 5 mM KCl, 1.3 mM CaCl2, 5 mM glucose, 1.5% [w/v] glucose, and 0.1% [w/v] collagenase I) for 20 minutes at 37°C with shaking. After incubation, the mixture was centrifuged, and the pelleted stromal vascular fractions (SVFs) were collected for flow cytometry analysis. SVFs were separated from red blood cells by adding lysis buffer (155 mM NH4Cl/0.1 M Tris-HCl, pH 7.65, in a 9:1 ratio). Following washing with 1X PBS, SVFs were stained with monoclonal antibodies against CD11b (1:250, BD Pharmingen, San Diego, CA, USA, #557396), F4/80 (1:100, BD Horizon, Franklin Lakes, NJ, USA, #563900), CD11c (1:250, Invitrogen, #12-0114-81), and CD206 (1:250, BD Pharmingen, #565250) for macrophage analysis using a FACS Canto II (BD Biosciences).
Adipose tissue histology
Formalin-fixed paraffin-embedded epididymal adipose tissue samples were sectioned to 5 µm thickness, deparaffinized in xylene and then rehydrated using a gradient of alcohol before undergoing hematoxylin and eosin staining. Images were captured using a Leica bright-field microscope (Leica, Wetzlar, Germany) at a ×20 magnification. Adipocyte sizes were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA).
Immunostaining of hypothalamus
Mice were anesthetized with an intraperitoneal injection of 40 mg/kg Zoletil® (Virbac, Carros, France) and 5 mg/kg Rompun (Bayer AG, Leverkusen, Germany) and then perfused with 50 mL saline followed by 50 mL 4% paraformaldehyde (PFA) via the left ventricle of the heart. The obtained brains were post-fixed with 4% PFA for 16 hours at 4°C and then dehydrated in a 30% sucrose solution until the tissues sank to the bottom of the container. Coronal brains, including the hypothalamic area (bregma –1.22 to –2.30 mm), were sectioned at a thickness of 30 µm using a cryostat (Leica). One out of every five slides was collected and measured. For Iba1 and glial fibrillary acidic protein (GFAP) staining, hypothalamic slices were blocked with 3% donkey serum for 1 hour and then incubated with Iba1 (1:400, goat, Abcam, Cambridge, UK, #5076) and GFAP (1:1,000, rabbit, Millipore, Burlington, MA, USA, #AB5804) at 4°C for 48 hours. For microtubule associated protein 2 (MAP2) and green fluorescence protein (GFP) staining, hypothalamic slices were permeabilized in 0.5% phosphate buffered saline-tween 20 (PBST) for 5 minutes, blocked with 3% donkey serum for 1 hour, and then incubated with MAP2 (1:1,000, Chicken, Abcam, #5392) and GFP (1:1,000, rabbit, Abcam, #6556) antibodies at 4°C overnight. After washing, slides were incubated with Alexa Fluor-conjugated secondary antibodies (1:1,000, Invitrogen) at room temperature for 2 hours. For nuclear staining, slides were treated with 4’,6-diamidino-2-phenylindole (DAPI) (5 mg/mL, 1:10,000, Sigma, #20718-90-3) for 10 minutes before mounting. Immunofluorescence images were obtained using a confocal microscope (Carl Zeiss 710, Oberkochen, Germany).
Intracerebroventricular leptin injection
A stainless steel cannula (26 gauge) was implanted into the third ventricle (3V) of the mice using stereotaxic coordinates: 1.4 mm caudal to the bregma and 5.5 mm ventral to the sagittal sinus. Following a 7-day recovery period, the correct positioning of each cannula was confirmed by a positive dipsogenic response from administration of 50 ng of angiotensin-II. Animals with a negative drinking response to angiotensin-II were excluded from the study.
Insulin and leptin signaling
To evaluate insulin signaling in the adipose tissues, either insulin (Humulin-R 0.25 U/kg) or saline was administered intraperitoneally to mice receiving an HFD for 8 weeks following an overnight fast. Fifteen minutes after injection, epididymal adipose tissues were collected for Akt phosphorylation analysis. To assess leptin signaling in the hypothalamus, leptin (1 μg) or saline was injected intracerebroventricularly following an overnight fast in 8-week HFD fed mice. Food intake was monitored for 24 hours after intracerebroventricular (ICV) injections. Three days later, animals were sacrificed 45 minutes after the ICV injections of leptin and mediobasal hypothalamic blocks were collected for analysis of signal transducer and activator of transcription 3 (STAT3) phosphorylation.
Gene expression
Mice were kept under freely-fed conditions and sacrificed from 9:00 AM to 10:00 AM. Mediobasal hypothalamus (MBH), brown adipose tissue (BAT), and both inguinal and epididymal white adipose tissue (WAT) were then harvested, snap frozen in liquid nitrogen, and stored at –70°C. Total RNA was subsequently extracted from these tissues using TRIzol in accordance with the manufacturer’s protocol. 5 μg RNA aliquots were then reverse transcribed to generate cDNA. The threshold cycle (CT) values were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers used for qPCR analysis are listed in Supplementary Table 1.
Enzyme-linked immunosorbent assay
Plasma IL-6 levels (50 μL) were assayed using mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, #M6000B-1) following the manufacturer’s protocol.
Immunoblotting
Protein lysates extracted from the mouse MBH and adipose tissues (20 µg protein each) were subjected to immunoblotting. Briefly, following blocking in 3% skim milk for 1 hour at room temperature, the membranes were incubated overnight at 4°C with primary antibodies targeting total STAT3 (1:1,000, Cell Signaling, Danvers, MA, USA, #4904S), phospho (Y705)-STAT3 (1:1,000, Cell Signaling, #9145S), total Akt (1:1,000, Cell Signaling, #9272S), phospho (Ser473)-Akt (1:1,000, Cell Signaling, #9271S), and β-actin (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA, #sc-47778).
Statistical analysis
All data are presented as the mean±standard error of the mean. Statistical analyses were performed using Prism 9.1.2 (GraphPad Software Inc., San Diego, CA, USA). Cell numbers and immunofluorescence intensities were quantified using Image J and Photoshop version CS6 (Adobe Systems, San Jose, CA, USA). Statistical significance among the study groups was tested using one-way or two-way analysis of variance (ANOVA) followed by a post hoc Tukey test or an unpaired Student’s t-test if appropriate. Analysis of covariance (ANCOVA) (SPSS version 28, IBM Co., Armonk, NY, USA) was used to compare the group’s EE. Statistical significance was defined as P<0.05.
Ethics statement
All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC, #2015-11-214) of the Asan Institute for Life Sciences (Seoul, Korea).
RESULTS
Generation of mice with a macrophage Grn deficiency
To investigate the metabolic roles of macrophage-derived PGRN, we generated a mouse model in which the PGRN-encoding gene, Grn, was specifically depleted in bone marrow-derived macrophages. We achieved this by crossbreeding mice expressing Cre recombinase under the control of the LysM promoter (LysM-Cre mice) with Grn-floxed mice, resulting in mice referred to as Grn-MΦKO. To verify the Grn deletion in macrophages, we labeled blood myeloid cells with tdTomato by mating LysM-Cre mice or Grn-MΦKO mice with tdTomato reporter mice. tdTomato-labeled blood myeloid cells were isolated using flow cytometry (Supplementary Fig. 1A) and sorted into PGRN+ cells or PGRN− cells. The PGRN+ cell population was reduced in the Grn-MΦKO;tdTomato mice compared to the LysM-Cre;tdTomato mice (Supplementary Fig. 1B). We further found that the Grn expression level in CD11b+ Ly6C+ blood monocytes was significantly lower in Grn-MΦKO mice than in Grn-WT mice (Supplementary Fig. 1C), thus confirming the successful depletion of macrophage Grn.
Some neurons have been shown to be labeled in LysM-Cre reporter mice [21] To address this concern, we generated LysM-Cre-eGFP mice and tested the eGFP expression in hypothalamic neurons. The colocalization of eGFP and neuronal marker MAP2 expression was very low in the ARC, paraventricular nucleus, and dorsomedial nucleus (Supplementary Fig. 2) indicating that hypothalamic neuronal Grn depletion was minimal in the Grn-MΦKO mice.
A macrophage Grn deficiency in mice does not alter energy or glucose metabolism under normal dietary conditions
We next conducted metabolic phenotype analysis in Grn-MΦKO male mice in comparison with Grn-WT mice under NCD-fed conditions. No significant differences were found in body weight, fat mass, or lean mass levels between the two groups of mice (Fig. 1A and B). Consistently, monitoring of food intake and EE revealed no changes associated with macrophage Grn depletion (Fig. 1C and D). The respiratory exchange ratio (RER), an index of nutrient oxidation for fuel, was also unchanged in NCD-fed Grn-MΦKO mice (Fig. 1E). There was no detectable alteration in locomotor activity (Supplementary Fig. 3). We further evaluated glucose metabolism by conducting GTT and ITT. Changes in blood glucose levels after the glucose and insulin challenges did not differ between the two groups (Fig. 1F and G). These data implied that a PGRN deficiency in macrophages does not significantly affect energy and glucose metabolism under normal diet conditions.
Macrophage Grn deficiency does not cause alterations to energy or glucose metabolism under normal diet conditions. (A, B) Comparison of body weights and fat and lean masses between normal chow diet (NCD)-fed Grn-wild-type (WT) (Grnf/f) mice and macrophage-specific Grn depletion (Grn-MΦKO) mice (n=4). (C) Average daily food intake values from 5 to 20 weeks of age (n=4). (D) Energy expenditure (EE) curves and average values during the day and night measured at 15 weeks of age (n=4). (E) Respiratory exchange ratio (RER) measured at 15 weeks of age (n=4). (F, G) Blood glucose levels and area under the curve (AUC) values during glucose and insulin tolerance tests (GTT and ITT) conducted at 14 weeks of age (n=4). Results are presented as a mean±standard error of the mean. NS, not significant.
A macrophage Grn deficiency prevents obesity and improves glucose metabolism under high-fat diet conditions
We next investigated the metabolic phenotype of Grn-MΦKO mice under conditions of overnutrition. These mice were fed an HFD starting at 7 weeks of age until sacrifice. Grn-MΦKO mice gained less weight during this HFD challenge compared to wild littermates (Fig. 2A). The lower body weights of the Grn-MΦKO mice were due to a lower fat mass, as their lean mass was unchanged (Fig. 2B) and demonstrated a resistance to DIO. We further found that this obesity resistance was attributable to an enhanced EE, especially during nighttime, with no alterations in food intake and RER (Fig. 2C and D, Supplementary Fig. 4A). Locomotor activity was not significantly altered in the HFD-fed Grn-MΦKO mice compared with their diet-matched wild littermates (Supplementary Fig. 4B). We thus explored the possibility that increased EE might be caused by changes in thermogenesis. Indeed, The mRNA expression levels of thermogenic genes such as uncoupling protein 1 (Ucp1), PR domain containing 16 (Prdm16), and iodothyronine deiodinase 2 (Dio2) were increased in the BAT and inguinal WAT of Grn-MΦKO mice (Fig. 2E and F).
Macrophage Grn deficiency prevents obesity and improves glucose metabolism under high-fat diet (HFD) conditions. (A, B) Body weights and fat and lean masses in HFD-fed Grn-wild-type (WT) (Grnf/f) mice and macrophage-specific Grn depletion (Grn-MΦKO) mice (n=5). (C) Average daily food intake from 5 to 20 weeks of ages (n=5). (D) Energy expenditure (EE) in Grn-WT (Grnf/f) mice and Grn-MΦKO mice fed an HFD for 15 weeks (n=4). (E, F) mRNA expression levels of thermogenic genes in the brown adipose tissue (BAT) and inguinal white adipose tissues (iWAT) of HFD-fed Grn-WT and Grn-MΦKO mice (n=4). (G, H) Glucose and insulin tolerance tests (GTT and ITT) conducted after 14 weeks of HFD feeding (n=5). Results are presented as a mean±standard error of the mean. NS, not significant; Ucp1, uncoupling protein 1; Prdm16, PR domain containing 16; Pgc1α, Pparg coactivator 1 alpha; Dio2, iodothyronine deiodinase 2; AUC, area under the curve. aP<0.05, bP<0.01, cP<0.001 between indicated groups.
Glucose and insulin tolerance were also improved in HFD-fed Grn-MΦKO mice compared to the diet-matched wild controls (Fig. 2G and H). These findings suggested that a macrophage Grn deficiency protects against HFD-induced obesity and its metabolic complications.
HFD-induced changes in adipose tissues are fully suppressed by a macrophage Grn deficiency
As changes in the WAT are closely related to the development of insulin resistance and glucose intolerance under DIO conditions [2,3], we examined whether any WAT changes arose in the Grn-MΦKO mice upon HFD challenge. Histological analysis of epididymal WAT revealed that HFD feeding increased adipocyte sizes in Grn-WT mice, but that these HFD-induced changes were blunted in the Grn-MΦKO mice (Fig. 3A). Crown-like structures (CLS) in WAT are histological features characterized by macrophages surrounding dead or dying adipocytes [22], that indicate adipose tissue inflammation in conditions of obesity. As expected, CLS formation in WAT increased in HFD-fed Grn-WT mice compared to NCD-fed Grn-WT mice. Notably, HFD-induced CLS formation was reduced in Grn-MΦKO mice to the levels of NCD-fed Grn-WT mice (Fig. 3A). These histological findings suggested that Grn depletion in macrophages prevents adipocyte hypertrophy and ATM accumulation under conditions of chronic overnutrition.
High-fat diet (HFD)-induced changes in the adipose tissues are fully suppressed by macrophage Grn deficiency. (A) Histological analysis of the epididymal while adipose tissues of normal chow diet (NCD)-fed Grn-wild-type (WT) mice and HFD (10 weeks)-fed Grn-WT and macrophage-specific Grn depletion (Grn-MΦKO) mice. Measurements of adipocyte sizes and numbers of crown-like structure (CLS) formations (n=6). Scale bars, 200 μm. (B) Fluorescence-activated cell sorting analysis of stromal vascular fractions of the epididymal adipose tissues showing the M1 and M2 polarization of adipose tissue macrophages (ATMs) in NCDPGRN+or HFD (10 weeks)-fed Grn-WT and HFD-fed Grn-MΦKO mice (n=3–4). (C) Quantitative polymerase chain reaction analysis of inguinal white adipose tissues (n=6). (D) Enzyme-linked immunosorbent assay (ELISA) of plasma interleukin 6 (IL-6) levels (n=4–7). (E) Immunoblotting analysis of Akt phosphorylation in the adipose tissue in response to intraperitoneal insulin injection (n=3–4). Results are presented as a mean±standard error of the mean. C/ebpα, CCAAT enhancer binding protein α; Pparγ, peroxisome proliferator-activated receptor γ; Fabp4, fatty acid binding protein 4; Glut4, glucose transporter 4; Tnfα, tumor necrosis factor-α; Ccl2, C-C motif chemokine ligand 2; NS, not significant. aP<0.05, bP<0.01, cP<0.001 between indicated groups.
Macrophages are categorized into two main polarization types: classically-activated (M1) and alternatively-activated (M2) [4,23]. During the progression of DIO, M1 macrophages increase while M2 macrophages decrease in the adipose tissues, contributing to adipose tissue inflammation and the development of insulin resistance [4]. We therefore investigated changes in macrophage polarization in our mouse models. Flow cytometry analysis of SVFs, obtained from epididymal adipose tissues, revealed that among the total ATMs (CD11b+ F4/80+), the proportion of M1 cells (CD11c+) was elevated, while the proportion of M2 cells (CD206+) was reduced in HFD-fed Grn-WT mice compared to NCD-fed Grn-WT mice (Fig. 3B). Strikingly, these HFD-induced changes in macrophage polarization were completely prevented in the HFD-fed Grn-MΦKO mice (Fig. 3B). Consistently, the M1/M2 ratio in adipose tissues was increased by HFD feeding, but this change was fully suppressed by a macrophage Grn deficiency (Fig. 3B). These findings suggested that macrophage PGRN may play a critical role in ATM polarization during chronic HFD consumption.
Quantitative PCR analysis of inflammation-related genes further revealed that HFD-fed Grn-MΦKO mice exhibited a lower expression of proinflammatory cytokines (Il-6, Tnfα) and the chemokine C-C motif chemokine ligand 2 (Ccl2), along with increased expression of the anti-inflammatory cytokine Il-10, reflecting reduced adipose tissue inflammation (Fig. 3C). In addition, the mRNA expression levels of peroxisome proliferator-activated receptor γ (Pparγ), a key transcription factor in adipogenesis, and the adipocyte-derived hormone leptin were reduced in HFD-fed Grn-MΦKO mice (Fig. 3C). These changes were consistent with the observed reduction in adipose mass. A previous study has reported that a generalized Grn depletion blocked an HFD-induced elevation in plasma and adipose tissue IL-6 expression levels [17]. Likewise, we found that plasma IL-6 levels were elevated by HFD consumption in Grn-WT mice but that this increase was significantly blocked in Grn-MΦKO mice (Fig. 3D).
We further investigated adipose tissue insulin signaling, given that HFD-fed Grn-MΦKO mice showed improved insulin resistance (Fig. 2H). Insulin-induced Akt phosphorylation in epididymal WAT was reduced in HFD-fed Grn-WT mice compared to their NCD-fed counterparts, but recovered in HFD-fed Grn-MΦ mice (Fig. 3E). These findings suggest that a macrophage-specific Grn deficiency is sufficient to rescue HFD-induced insulin resistance in adipose tissue.
Reduced inflammation and improved leptin sensitivity in the hypothalamus of macrophage Grn-deficient mice
As hypothalamic macrophages are activated to sustain hypothalamic inflammation and disrupt metabolic hormonal signaling under HFD conditions [9], we tested for alterations in hypothalamic macrophages in HFD-fed Grn-MΦKO mice in comparison with NCDPGRN+and HFD-fed Grn-WT mice. We traced these cells by cross-bleeding of LysM-Cre mice or Grn-MΦKO mice with Cre-dependent GFP reporter mice. Consistent with the findings of a previous study [9], we found that 10 weeks of HFD feeding significantly increased the numbers of GFP+ hypothalamic macrophages in the ARC of LysM-Cre;GFP mice (Fig. 4A). In addition, the HFD conditions caused morphological changes in macrophages from a rod shape without cellular processes to an enlarged soma with multiple processes (Fig. 4A). The macrophage numbers and morphological changes in the HFD-fed Grn-MΦKO mice were remarkably attenuated, indicating less macrophage activation in these animals (Fig. 4A). We further examined changes in the Iba1+ hypothalamic microglia and GFAP+ astrocytes as their activation is a cardinal feature of hypothalamic inflammation in DIO mice [10]. HFD-induced increases in microglia and astrocyte numbers were significantly reduced in Grn-MΦKO mice on HFD feeding for 10 weeks (Fig. 4B and C). The soma sizes of Iba1+ microglia showed a decrease (Fig. 4B). These findings suggested a reduction in hypothalamic microglia and astrocyte activation in Grn-MΦKO mice despite consuming a similar amount of HFD to the controls.
Reduced inflammation and improved leptin sensitivity in the hypothalamus of macrophage Grn-deficient mice. (A) Green fluorescence protein (GFP) immunostaining showing GFP-labeled lysozyme M (LysM)+ macrophages in the hypothalamic arcuate nucleus (ARC) of LysM-Cre;GFP mice on an normal chow diet (NCD) and LysM-Cre;GFP and macrophage-specific Grn depletion (Grn-MΦKO);GFP mice on an high-fat diet (HFD) for 10 weeks (n=4). Scale bars, 50 μm. (B) Ionized calcium-binding adaptor molecule 1 (Iba1) immunostaining showing changes in the hypothalamic microglia (n=4). Scale bars, 50 μm. (C) Glial fibrillary acidic protein (GFAP) immunostaining showing changes in the hypothalamic astrocytes (n=4). Scale bars, 50 μm. (D) Quantitative polymerase chain reaction analysis of proinflammatory cytokines (interleukin 1β [Il-1β], Il-6, tumor necrosis factor-α [Tnfα]) and anti-inflammatory cytokines (Il-4, Il-10) in the mediobasal hypothalamus under 10-week HFD-fed conditions (n=6). (E) Food intakes for 24 hours after the intracerebroventricular administration of leptin (1 μg) in 8-week HFD-fed mice (n=4–5). (F) Determination of hypothalamic leptin signaling via the immunoblotting of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and total STAT3 at 45 minutes following the intracerebroventricular injection of saline or leptin (n=3). Results are presented as a mean±standard error of the mean. NS, not significant. aP<0.05, bP<0.01, cP<0.001 between indicated groups.
Analysis of proinflammatory and anti-inflammatory cytokine expression in the hypothalamus revealed that chronic HFD consumption upregulated the Il-1β, Il-6, and Tnfα expression levels but downregulated Il-4 expression (Fig. 4D). Notably HFD-induced elevation in proinflammatory cytokine expression was completely reversed in the hypothalamus of Grn-MΦKO mice (Fig. 4D). These data suggested that macrophage PGRN actively participates in the hypothalamic inflammation triggered by fat-rich diets.
Leptin, a critical hormone for maintaining energy homeostasis, exerts its effects primarily on the hypothalamus [24]. We therefore investigated whether a macrophage Grn deficiency could influence hypothalamic leptin signaling and its anorexigenic actions. ICV leptin injection suppressed food intake for 24 hours in NCD-fed Grn-WT mice and the anorexigenic effect of leptin was blunted in 8-week HFD fed Grn-WT mice, suggesting the development of hypothalamic leptin resistance following chronic HFD consumption (Fig. 4E). Notably, Grn-MΦKO mice on an HFD for 8 weeks exhibited improved feeding response to leptin compared to diet-matched Grn-WT mice (Fig. 4E). We finally assessed leptin signaling by measuring hypothalamic STAT3 phosphorylation. Leptin-induced STAT3 phosphorylation in the hypothalamus was significantly reduced in 8-week HFD fed Grn-WT mice compared to NCD-fed Grn-WT mice (Fig. 4F). In contrast, the Grn-MΦKO mice fed HFD for the same duration exhibited improved STAT3 phosphorylation in response to leptin (Fig. 4F). Taken together, these findings indicated that a lack of Grn in hypothalamic macrophages protects against hypothalamic leptin resistance under the conditions of fat overconsumption.
DISCUSSION
Our present findings in a mouse model have shown that a specific Grn depletion in macrophages affects metabolism in a diet-dependent manner. A lack of macrophage Grn has no apparent effect on adiposity or glucose metabolism in NCD-fed lean mice. In contrast, this macrophage Grn deficiency attenuates the development of obesity and glucose deregulation during chronic HFD feeding. Notably, macrophage-restricted Grn depletion produces similar metabolic outcomes to those seen in Grn−/− mice [17]. These results suggest that macrophage-derived PGRN may play a predominant role in promoting obesity development and its metabolic complications under conditions of overnutrition.
Our study has further revealed that the reduced inflammation and macrophage activation in the visceral adipose tissues and hypothalamus of Grn-MΦKO mice may underlie the role of PGRN to obesity. Notably, Grn depletion in ATMs was found to robustly suppress HFD-induced increases in CLSs in the gonadal WAT, a phenomenon similarly observed in Grn−/− mice [17]. Conversely, treatment with recombinant human PGRN increases monocyte chemotaxis [25]. Moreover, circulating blood PGRN levels are significantly correlated with macrophage infiltration in omental adipose tissues and the body mass index [25]. These findings collectively support the idea that macrophage PGRN facilitates macrophage infiltration into adipose tissues during the development of obesity.
We further found that macrophage PGRN may have a regulatory role in ATM polarization. As reported previously [4], chronic HFD feeding increased the M1 macrophage levels and decreased the number of M2 macrophages in our Grn-WT mice. However, a lack of Grn expression in macrophages reversed the HFD-induced polarization of ATMs toward M1. In contrast to our present findings, PGRN has been shown to inhibit lipopolysaccharide-induced macrophage M1 polarization [26]. The mechanisms underlying this contrasting effect of PGRN on macrophage polarization remain to be elucidated.
Similar to its effect in the adipose tissue, macrophage Grn depletion prevented perivascular macrophage activation and accumulation in the hypothalamic ARC. This result indicated an involvement of macrophage-derived PGRN in promoting HFD-induced macrophage activation in the hypothalamus. Unlike ATMs, hypothalamic macrophages locally proliferate and maintain their numbers without the recruitment of circulating monocytes, even under conditions of chronic HFD feeding [27]. Thus, we speculate that PGRN may regulate the pools of hypothalamic perivascular macrophages through local proliferation. Activated perivascular macrophages trigger the activation of other glial cell populations, such as microglia and astrocytes, a phenomenon commonly observed in HFD-induced hypothalamic inflammation [6,9,10]. Consistently, Grn-MΦKO mice exhibited less activation of microglia and astrocytes along with reduced proinflammatory cytokine expression in the hypothalamus. Overall, our current findings support the proinflammatory role of hypothalamic macrophage PGRN in the context of DIO.
Intriguingly, many previous studies using Grn knockout mice have reported conflicting results. Grn-deficient macrophages release a lower level of anti-inflammatory IL-10 and a higher amount of proinflammatory cytokines [28]. These cells have also been shown to be cytotoxic to neurons [28]. That same study also reported that Grn-deficient mice displayed greater activation of microglia and astrocytes with age [28]. In line with this, PGRN is expressed in macrophages in atherosclerotic lesions. Generalized Grn depletion aggravates atherosclerosis in apolipoprotein E knockout mice with increased expression of inflammatory cytokines and adhesion molecules [29]. Taken together, these findings support the anti-inflammatory effects of PGRN or other Grn products. In contrast, another prior study reported that Grn depletion in hematopoietic cells protects atherosclerosis in low-density lipoprotein-receptor knockout mice, indicating a proatherogenic effect of PGRN [30]. These results imply differential roles for PGRN or other Grn products with respect to inflammatory processes, depending on the released cell types and inflammatory contexts. Another potential mechanism underlying the contrasting outcomes of Grn deficiency may be related to the proteolytic processing of PGRN into GRNs with different biological functions [14-16,31]. Indeed, it is reported that PGRN and GRNs have opposing effects on wound healing [16].
The HFD-fed Grn-MΦKO mice in our present experiments exhibited enhanced Akt phosphorylation in visceral adipose tissues in response to insulin. This suggested that macrophage PGRN or Grn derived peptides may impede insulin sensitivity in the adipocytes of DIO mice. Likewise, macrophage PGRN seems to hinder leptin sensitivity in hypothalamic neurons. Macrophage PGRN may indirectly influence insulin or leptin signaling by regulating the release of proinflammatory molecules by macrophages. Alternatively, macrophage-secreted PGRN might enter neighboring adipocytes or neurons via sortilin-dependent or -independent mechanisms [32,33], potentially affecting insulin and leptin signaling pathways. Further investigations are needed to elucidate the detailed mechanisms by which PGRN modulates insulin and leptin signaling. Despite increased hypothalamic leptin signaling and a heightened anorexic response to leptin, Grn-MΦKO mice consumed amounts of food comparable to those of WT controls under freely-fed condition. Typically, when EE increases, animals adjust by increasing their food intake to maintain energy balance. Therefore, given the higher EE in Grn-MΦKO mice, the unchanged food intake could actually indicate a lower relative food intake.
In summary, our study identifies macrophage-derived PGRN as an important contributor to metabolic inflammation and deregulation during the progression of DIO. Given the complexity of Grn-derived peptides and their distinct roles, further studies are needed to clarify whether other Grn products may also be associated with the anti-obesity and anti-inflammatory phenotypes observed in Grn-MΦKO mice.
SUPPLEMENTARY MATERIALS
Supplementary materials related to this article can be found online at https://doi.org/10.4093/dmj.2024.0486.
Primer sequences used for quantitative polymerase chain reaction analysis
Confirmation of successful progranulin (PGRN) deletion in lysozyme M (LysM)+ monocytes. (A) Fluorecence-activated cell sorting (FACS) gating strategy of fluorescent intensity between C57BL6/J and LysM-Cre;tandem dimer tomato (tdTomato) mice. (B) FACS analysis indicates a significant reduction in the number of PGRN+ tdTomato+ blood myeloid cells in macrophage-specific Grn depletion (Grn-MΦKO);tdTomato mice (n=4). (C) FACS sorting of CD11b+, lymphocyte antigen 6 family member C (Ly6C)+ blood monocytes and comparison of Grn mRNA expression between Grn-wild-type (WT) and Grn-MΦKO mice (n=4–5). Results are presented as a mean±standard error of mean. aP<0.05, bP<0.01 between indicated groups.
Double immunohistochemistry of neuronal marker microtubule associated protein 2 (MAP2) and green fluorescence protein (GFP) in hypothalamic arcuate nucleus (ARC), paraventricular nucleus (PVH), and dosomedial nucleus (DMH) of high-fat diet fed lysozyme M (LysM)-GFP mice. Scale bars, 50 μm. DAPI, 4´,6-diamidino-2-phenylindole.
Unaltered locomotor activity in normal chow diet (NCD)-fed macrophage-specific Grn depletion (Grn-MΦKO) mice. Locomotor activity were measured using comprehensive lab monitoring system (CLAMS) in 15 weeks-old Grn-wild-type (WT) and Grn-MΦKO mice on a NCD (n=4). NS, not significant.
Unaltered respiratory exchange ratio (RER) (A) and locomotor activity (B) in high-fat diet (HFD)-fed macrophage-specific Grn depletion (Grn-MΦKO) mice. RER and locomotor activity were measured using comprehensive lab monitoring system (CLAMS) in 21-week-old Grn-wild-type (WT) and Grn-MΦKO mice on a HFD for 15 weeks (n=4). NS, not significant.
Notes
CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was reported.
AUTHOR CONTRIBUTIONS
Conception or design: C.H.L., J.B.K., M.S.K.
Acquisition, analysis, or interpretation of data: C.H.L., C.B.P., H.K.K., W.H.J., S.H.M.
Drafting the work or revising: C.H.L., M.S.K.
Final approval of the manuscript: M.S.K.
FUNDING
This study was supported by grants from the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT of Korea (2020R1A2C3004843, 2022M3E5E8017213, 2022R1C1C1012590, 2022R1C1C1004187), and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (RS-2024-00438349, RS-2024-00404132). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
ACKNOWLEDGMENTS
We thank Boston BioEdit for help with preparing the manuscript.