Phosphodiesterase 5 Inhibitor Improves Insulin Sensitivity by Regulating Adipose Tissue Macrophage Polarization in Diet-Induced Obese Mice
Article information
Abstract
Background
Obesity is a rapidly increasing global health issue, which is associated with glucose and insulin resistance. Phosphodiesterase type 5 (PDE5) inhibitors (PDE5i) are known for their ability to enhance blood flow and vascular stability and are widely used to treat conditions such as erectile dysfunction, pulmonary hypertension, heart failure, and cancer. However, studies investigating the role of PDE5i in alleviating obesity and metabolic diseases remains unclear. Therefore, we investigated the effects of PDE5i on obesity and metabolic disorders in diet-induced obese mice and its underlying mechanisms.
Methods
PDE5i was administered to high-fat diet (HFD)-fed C57BL/6J mice for 6 to 7 weeks. Body weight and food intake were measured weekly, and baseline metabolic rates, physical activity, and glucose and insulin tolerance tests were assessed during PDE5i administration. Macrophages and T-cells in the gonadal white adipose tissue (gWAT) were analyzed by flow cytometry. Vascular stability and blood flow in gWAT were analyzed via immunostaining and in vivo live imaging. RAW264.7 cells and bone marrow-derived macrophages were used to determine immunoregulatory effects of PDE5i.
Results
In HFD-fed mice, PDE5i administration significantly enhanced systemic insulin sensitivity and AKT phosphorylation in gWAT. PDE5i reduced the M1/M2 ratio of gWAT macrophages of obese mice. These phenomena were associated with enhanced blood flow to the gWAT. In vitro experiments revealed that PDE5i suppressed lipopolysaccharide-induced proinflammatory cytokine production and increased the mRNA expression of genes associated with M2 polarization.
Conclusion
PDE5i plays a role in regulating adipose tissue inflammation and thus holds promise as a therapeutic agent for metabolic enhancement.
Highlights
• PDE5i administration improves insulin sensitivity without affecting body weight.
• PDE5i promotes M2 macrophage polarization and increase Tregs in gWAT.
• PDE5i enhances vascular stability and blood perfusion in gWAT of obese mice.
• PDE5i directly suppresses inflammation and induces M2 polarization in BMDMs.
INTRODUCTION
Obesity is a global pandemic characterized by low-grade systemic inflammation [1]. Low-grade inflammation, which is associated with metabolic disorders such as glucose intolerance and insulin resistance. These conditions can lead to the development of more serious diseases such as type 2 diabetes mellitus, cancer, and cardiovascular diseases [2]. Insulin resistance is closely associated with white adipose tissue inflammation [3-5]. Notably, the gene expression of proinflammatory cytokines, such as interleukin 1β (Il1β) and tumor necrosis factor-α (Tnfα), is upregulated in the gonadal white adipose tissue (gWAT) of mice fed a high-fat diet (HFD) compared to that in mice fed a chow diet (CD) [6].
Adipose tissue macrophages (ATMs) play an important role in adipose tissue inflammation [6]. ATMs have heterogeneous characteristics and are classified into classically activated ATMs (M1 ATMs) and alternatively activated ATMs (M2 ATMs), distinguished by the surface markers CD11c and CD206, respectively [7-9]. The M1/M2 ratio in adipose tissue is closely associated with the degree of HFD-induced insulin resistance [10]. ATM polarization may be associated with T-cell activity [11]. CD4 T-cells, including T helper 1 (Th1), Th2, and regulatory T (Treg) cells, communicate with ATMs, influencing the immune response in adipose tissue. Th1 cells induce M1 ATM polarization by secreting interferon-γ (IFNγ), whereas Th2 cells induce M2 ATM polarization by secreting IL-4 and IL-13 [12-15]. Treg cells, which exhibit immunoregulatory effects, can induce M2 ATM polarization through IL-10 signaling, thereby enhancing insulin sensitivity [16]. Therefore, research targeting immune cell regulation aimed to improve metabolism in adipose tissue is essential.
Cyclic guanosine monophosphate (cGMP) reduces blood pressure by enhancing endothelial cell permeability and decreasing blood vessel constriction [17]. The enzyme phosphodiesterase type 5 (PDE5) limits vascular relaxation by breaking down cGMP, and it inhibition of PDE5 results in vasodilation and improved blood circulation [18]. Consequently, PDE5 inhibitors (PDE5i) have been widely used to treat erectile dysfunction. Owing to its role in vasodilation and blood flow enhancement, PDE5i has also been utilized in treating pulmonary hypertension (PH) [19,20] and cancers, including prostate, breast, and colon cancer [21]. PDE5 inhibition leads to significantly decreased pulmonary vascular resistance, thereby alleviating PH symptoms and improving exercise tolerance in patients with PH [20]. Moreover, co-administration of the chemotherapeutic anticancer drugs sildenafil with doxorubicin enhances drug delivery, resulting in reduced breast cancer tumor sizes, compared to doxorubicin administration alone [21,22]. The application of PDE5i for improving blood flow and blood vessel stabilization has been explored for various therapeutic purposes. However, limited studies have investigated its use in alleviating obesity and metabolic disorders, with only one report demonstrating that enhanced blood perfusion is associated with ameliorated metabolic disorders [23].
In the present study, we investigated whether PDE5i can overcome obesity and metabolic disorders in HFD-fed obese mice and explored the role of vascular normalization and ATM polarization in this process. We also aimed to repurpose PDE5i, which has been validated for its safety and efficacy [24,25], thereby reducing the cost and time associated with drug development for metabolic diseases.
METHODS
Mice
Seven-week-old C57BL/6J male mice were purchased from DBL (Eumseong, Korea). Mice were individually housed in cages in a temperature-controlled room (22°C±1°C) with a 12-hour light-dark cycle (lights on 8:00 AM). Mice were provided with either CD (Cargill Agri Purina, Seongnam, Korea, #EEGJ30060) or 60% HFD (Research Diet, New Brunswick, NJ, USA, #D12492) and water ad libitum. Body weight and food intake were monitored daily or weekly. All animal experiments were approved by the Institutional Animal Care and Use Committee of Hallym University (Hallym 2024-9).
Drugs and experimental design
To evaluate the effects of PDE5i on obesity and metabolic disorders in diet-induced obese (DIO) mice, 5 or 10 mg/kg of PDE5i was orally administered daily for 6–7 weeks to mice fed an HFD for 4 weeks. The PDE5i sildenafil was purchased from MedChemExpress (Monmouth Junction, NJ, USA, #HY-15025) and dissolved in corn oil (Sigma-Aldrich, St. Louis, MO, USA, #C8267) containing 1% dimethyl sulfoxide (DMSO; Duchefa, Haarlem, The Netherlands, #D1370). The administration doses were determined based on previous reports [26,27]. PDE5i was administered daily between 9:00 AM and 10:00 AM, and body weight and food intake were measured weekly between 9:00 AM and 10:00 AM. Oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPITT) were conducted between weeks 5 and 7 after PDE5i administration, while flow cytometry (FACS) analysis, histological, and adipose tissue insulin sensitivity analyses were performed at week 6 after PDE5i administration. For the signal transducer and activator of transcription 6 (STAT6) inhibition experiments, mice were divided into HFD+vehicle, HFD+PDE5i, and HFD+PDE5i+AS-1517499 groups. The STAT6 phosphorylation in hibitor AS-1517499 (Sigma-Aldrich, #HY-100614) was administered intraperitoneally every other day for 2 weeks before euthanasia. AS-1517499 was dissolved in 10% DMSO in corn oil, while the vehicle comprised 10% DMSO in corn oil. Insulin sensitivity and immune cell polarization in gWAT were examined via IPITT and FACS.
Metabolic analysis
For baseline metabolic analysis, the Comprehensive Lab Animal Monitoring System (CLAMS; PhenoMaster, TSE systems, Berlin, Germany) was used to measure parameters including O2 consumption (VO2), CO2 production (VCO2), energy expenditure, respiratory exchange ratio (RER), and locomotor activity. HFD-fed mice received vehicle or PDE5is at doses of 5 or 10 mg/kg and were placed in the metabolic chamber for 2 days of acclimate with data collected following 2 days. Average energy expenditure was calculated after correcting for body weight and analyzed using one-way analysis of covariance (ANCOVA). Total movement was quantified to assess locomotor activity. For the OGTT, glucose (1 g/kg; Sigma-Aldrich, #G8270) was administered orally to mice after overnight fasting. For the IPITT, insulin (Humulin-R 0.25 U/kg, Eli Lilly, Indianapolis, IN, USA, #170131BIJ) was intraperitoneally injected into overnight-fasted mice. Blood samples were obtained from the tail vein using a glucometer (ACCU-CHEK, Aviva Plus System, London, UK) at indicated time points after glucose or insulin injection. To assess insulin sensitivity in adipose tissues, 0.25 U/kg insulin was injected into the peritoneal cavity of mice administered with PDE5i for 6 weeks, and gonadal adipose tissues (gWAT) were obtained 10 minutes later.
FACS analysis
Adipose tissues, including gWAT, inguinal white adipose tissue (iWAT), and brown adipose tissue (BAT) were isolated from mice. Tissues were dissected and dissociated into single cells by chopping and digesting using an Adipose Tissue Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-105-808) and gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec, #130-096-427). Pelleted stromal vascular cell (SVC) fractions were separated from red blood cells using ammoniumchloride-potassium lysis buffer (150 mM NH4Cl, 1 M KHCO, and 0.5 M ethylenediaminetetraacetic acid [EDTA]). For transcription factor staining, SVCs were reactivated through incubation with Iscove’s Modified Dulbecco’s Medium (IMDM; supplemented with 10% fetal bovine serum [FBS] and 1% penicillin-streptomycin), followed by treatment with phorbol 12-myristate 13-acetate (Sigma-Aldrich, #P8139), ionomycin (Sigma-Aldrich, #I0634), and Golgi Stop (BD Biosciences, Franklin Lakes, NJ, USA, #554724) for 6 hours. SVCs were then incubated in an Fc blocker (BD Bioscience, #553142) with 7-aminoactinomycin D (1:62.5, Invitrogen, Waltham, MA, USA, #A1310). After washing with FACS wash buffer (1% FBS, 2 mM EDTA, and 0.05% NaN3), SVCs were incubated in a fixation/permeabilization buffer (Invitrogen, #00-5523-00) for 30 minutes and subsequently stained with monoclonal antibodies against cocktail #1 including CD4 (1:250, BD Pharmingen, San Diego, CA, USA, #553046), forkhead box P3 (FoxP3) (1:100, Invitrogen, #12-4771-82), IFNγ (1:250, BD Pharmingen, #554413), T-cell receptor β (TCRβ) (1:125, Invitrogen, #47-5961-82), CD25 (1:500, BD Horizon, Franklin Lakes, NJ, USA, #562606), and CD45 (1:250, BioLegend, San Diego, CA, USA, #103151). For intracellular staining, SVCs were blocked and then fixed in 4% paraformaldehyde (PFA; Tech&Innovation, Indianapolis, IN, USA, #BPP-9004) for 10 minutes. After washing with FACS wash buffer, SVCs were permeabilized with 0.2% saponin (Sigma-Aldrich, #47036) in FACS wash buffer for 10 minutes. They were then stained with cocktail #2 consisting of CD11b (1:250, BD Pharmingen, #557396), CD11c (1:250, Invitrogen, #12-0114-81), Ly-6G (1:200, BD Pharmingen, #560601), CD206 (1:250, BD Pharmingen, #565250), F4/80 (1:100, BD Horizon, #563900), and CD45 (1:250, BioLegend, #103151). To quantify cytokine production, a cytokine bead assay (CBA) was conducted in bone marrow-derived macrophages (BMDMs). Culture supernatants from BMDMs were quantified using the LEGENDplexTM Mouse Inflammation Panel (BioLegend, #740150). All procedures were performed following a previous study [28]. Data were analyzed using the FACSCanto II instrument (BD Biosciences) and the FlowJo software version 10.8 (https://www.flowjo.com).
Western blotting
gWAT tissues were homogenized in lysis buffer (Biosesang, Yongin, Korea, #RC2002-050-00) supplemented with protease inhibitors (GenDEPOT, Altair, TX, USA, #P3100), and phosphatase inhibitors (GenDEPOT, #P3200). Tissue lysates were centrifuged at 13,000 rpm at 4°C for 30 minutes. The supernatant was collected, and the protein concentration was measured then mixed with the sample buffer. Protein samples were separated on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and loaded onto a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA, #IPVH00010). The membranes were blocked with 3% skim milk (Carl Roth, Karlsruhe, Germany, #T145.1) or 5% bovine serum albumin (BSA; Bovogen, East Keilor, Australia, #BSAS 0.1) in 1X Tris buffered saline with Tween 20 (TBST) buffer (containing Tween 20, Tris, and NaCl) for 1 hour. Subsequently, the membranes were incubated at 4°C with primary antibodies against phospho AKT (1:1,000, Cell Signaling Technology [CST], Danvers, MA, USA, #9271), total AKT (1:1,000, CST, #9272), phosphor-4E-binding protein 1 (4EBP1) (1:3,000, CST, #2855), total 4EBP1 (1:3,000, CST, #9644), phospho S6 (1:3,000, CST, #5364), total S6 (1:3,000, CST, #2217), and β-actin (1:1,000, Santa Cruz, Santa Cruz, CA, USA, #47778) for 16 hours. After washing with 1X TBST buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature (RT) for 1 hour. Target proteins were visualized using a chemiluminescence imaging system (ImageQuant LAS 500, GE Healthcare, Chicago, IL, USA), and protein band were quantified using ImageJ version 1.8.0 (National Institutes of Health, Bethesda, MD, USA).
Immunohistochemistry
Mice were anesthetized with isoflurane (Hana Pharm Co. Ltd., Hwaseong, Korea) and then cardially perfused with 50 mL of pre-cooled saline, followed by 50 mL of pre-cooled 4% PFA. Whole mount staining of gWAT was performed following the procedure described by Xue et al. [29]. Briefly, adipose tissues were obtained and fixed in 4% PFA at 4°C for 16 hours. The tissues were digested by incubation with proteinase K (20 μg/mL, Cosmo Genetech, Seoul, Korea, #BP1700-100) in 10 mM Tris-HCl buffer at RT for 5 minutes. They were then permeabilized in methanol (Daejung, Siheung, Korea, #5558-4404) for 30 minutes and blocked with 3% skim milk dissolved in phosphate-buffered saline (PBS) with 0.3% Triton X-100 (PBST, Sigma-Aldrich, #T8787) at 4°C for 16 hours. Tissues were incubated with anti-mouse CD31 antibody (1:200, BD Pharmingen, #550274) in 3% blocking solution at 4°C for 16 hours. After washing with 1X PBS, the adipose tissues were incubated with Alexa-Flour 555-conjugated secondary antibody (1:400, Invitrogen, #A11081) at RT for 2 hours. For adherent junction staining, gWATs were embedded in paraffin and sectioned to a thickness of 5 μm. The slices were deparaffinized and then permeabilization with 0.5% PBST for 5 minutes at RT and blocked with 3% BSA dissolved in 0.5% PBST at RT for 1 hour. The slices were then treated with vascular endothelial-cadherin (VECad; 1:200, Santa Cruz, #sc9989) and CD31 (1:400, R&D system, Minneapolis, MN, USA, #AF3628) or neuron-glial antigen 2 (NG2; 1:200, Sigma-Aldrich, #AB5320) and CD31 (1:400) dissolved in PBS at 4°C for 16 hours, followed by incubation at RT for 1 hour. After washing with 1X PBS, the slices were incubated with the appropriate Alexa Fluor conjugated secondary antibody (1:1,000, Invitrogen, #A21432, #A21202, and #A21206) at RT for 1 hour. Immunofluorescence was imaged using a confocal microscope (Carl Zeiss, Oberkochen, Germany, #710).
Quantitative polymerase chain reaction analysis
Total RNA was extracted using the TRIzol reagent (Invitrogen, #15596018). cDNA was synthesized and quantitative polymerase chain reaction was conducted using SYBR green premix (Bioneer, Daejeon, Korea, #K6253) and appropriate primers (Supplementary Table 1). Quantitative analysis was performed using the ΔΔCT method, and mRNA expression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (Gapdh).
In vitro experiments
RAW264.7 cells were cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium (Welgene, Gyeongsan, Korea, # LM011-03) supplemented with 10% FBS (Gibco, Waltham, MA, USA, #12483-020) and 1% penicillin/streptomycin (P/S) (Gibco, #15140-122) in a humidified environment with 5% CO2. One the day before drug treatment, cells were seeded into a 6-well plate at a density of 5×105 cells/well. After washing with PBS, they were incubated with lipopolysaccharide (LPS; Sigma-Aldrich, #L2630) or PDE5i in a serum-free RPMI medium. Total RNA was extracted using the TRIzol reagent. Bone marrow cells were isolated from the mouse femur and tibia using IMDM media, and then cultured with macrophage colony-stimulating factor (M-CSF; Pepro Tech, Cranbury, NJ, USA, #315-02) for 1 week to induce their differentiation into BMDMs. Successful differentiation into BMDMs was confirmed via FACS analysis. For assessing M2 polarization of BMDMs induced by PDE5i, IL-4 was treated for 12 hours. For the Pde5 depletion study, scrambled shRNA or shPDE5a (Origene, Rockville, MD, USA, #TR515113) was transfected into BMDMs using Lipofectamine3000 (Invitrogen, #L3000015). Successful transfection was confirmed via FACS analysis. The transfected cells were subsequently treated with LPS, and cytokine production was measured using a CBA and FACS.
In vivo fluorescence live imaging
To assess blood flow in gWAT, we used an in vivo live imaging system (Vilber Lourmat, Marne-la-Vallée, France, NEWTON 7.0 #FT-100). After anesthesia, 0.001% Evans blue (Sigma-Aldrich, # E2129) was injected via the tail vein, and the fluorescence signal in gWAT was traced for 20 minutes. Fluorescence dye was excited at 620 nm using a Laser Class 2 LED (#C640) and detected at 680 nm using a hard-coated filter (#F700). Image capturing and analysis were conducted using the same imaging system.
Statistical analysis
All data are presented as mean±standard error of the mean. Statistical analyses were performed using the Prism software (GraphPad version 10.0; GraphPad Software Inc., San Diego, CA, USA). Statistical significance among groups was determined using one-way, two-way, or repeated-measures analysis of variance (ANOVA), followed by a post hoc least significant difference test. Energy expenditure was compared among the groups using one-way ANCOVA. Statistical significance was defined at P<0.05.
RESULTS
Chronic administration of PDE5i significantly improves insulin sensitivity without altering body weight and food intake in DIO mice
We investigated the effects of PDE5i in improving obesity and metabolic disorders in HFD-fed mice (Fig. 1A). We observed significant weight gain and insulin resistance in HFD-fed DIO mice compared to CD-fed mice (Supplementary Fig. 1). Single, short-term (7 days), and chronic administration (6 weeks) of PDE5i did not alter body weight or food intake (Fig. 1B-E, Supplementary Fig. 2A and B). We also conducted a CLAMS analysis to examine basal metabolism. Mice administered 10 mg/kg of PDE5i exhibited increased VO2, VCO2, and energy expenditure during the daytime, whereas 5 mg/kg of PDE5i did not elicit these changes (Fig. 1F-I). The RER was decreased during the daytime in mice administered with 10 mg/kg PDE5i compared to vehicle-administered mice (Fig. 1J). These findings suggested that PDE5i enhances energy metabolism during the daytime and increases fat utilization over carbohydrate consumption. Activity levels exhibited no significant differences during the measurements (Fig. 1K).
Phosphodiesterase type 5 (PDE5) inhibitor (PDE5i) administration does not alter body weight and food intake. (A) Experimental timetable of systemic PDE5i administration. (B, C) Changes in body weight and food intake following single administration of vehicle or PDE5i (n=4). (D, E) Changes in body weights and food intakes following chronic administration of vehicle or PDE5i (n=6). (F, G, H, I, J, K) Effects of PDE5i on the baseline metabolism (VO2, VCO2, energy expenditure, respiratory exchange rate, and locomotor activity) in high-fat diet (HFD)-fed obese mice (n=4). Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided one-way analysis of variance (ANOVA) (B, F, G, H, J, K), onesided two-way ANOVA (C, D, E), and one-way analysis of covariance (ANCOVA) using body mass as covariate (I). ITT, insulin tolerance test; CLAMS, Comprehensive Lab Animal Monitoring System; GTT, glucose tolerance test; NS, non-significant. aP<0.05, bP<0.001 between indicated groups.
We examined the effects of chronic PDE5i administration on systemic metabolic functions. The OGTT revealed showed that blood glucose levels followed a similar pattern in the vehicle, 5 mg/kg PDE5i, and 10 mg/kg PDE5i groups (Supplementary Fig. 3). However, administration of 10 mg/kg PDE5i for 4 and 6 weeks, but not that of 5 mg/kg PDE5i, significantly improved HFD-induced insulin resistance (Fig. 2A and B). Oneweek of PDE5i administration at a dose of 10 mg/kg resulted in a trend toward improved insulin sensitivity (Supplementary Fig. 2C). These results indicated that chronic PDE5i administration improved systemic insulin sensitivity.
Phosphodiesterase type 5 (PDE5) inhibitor (PDE5i) administration improves insulin sensitivity in high-fat diet (HFD)-induced obese mice. (A, B) Blood glucose levels measured by insulin tolerance tests performed after vehicle or PDE5i administration for 4 and 6 weeks (n=6). (C) Western blot data and quantification of phospho AKT, total AKT, phosphor-4E-binding protein 1 (4EBP1), total 4EBP1, phospho S6, total S6, and β-actin in gonadal white adipose tissue (gWAT) after insulin administration (n=6). Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided two-way analysis of variance (ANOVA) (A, B) or one-sided one-way ANOVA (C). IPITT, intraperitoneal insulin tolerance test; CD, chow diet. aP<0.05, bP<0.01, cP<0.001 between indicated groups.
In obese mice, an increase in body fat mass and metabolic disorders are closely related to insulin resistance in gWAT [30-32]. To confirm the correlation between gWAT and insulin sensitivity, we injected insulin into vehicle- or PDE5i-treated mice and isolated the gWATs. We subsequently examined the changes in AKT phosphorylation and the mammalian target of the rapamycin (mTOR) pathway, which are known to be associated with insulin sensitivity [33]. Mice fed an HFD for 10–11 weeks exhibited impaired AKT phosphorylation in gWAT in response to insulin injection (Fig. 2C). However, insulin-induced AKT phosphorylation was significantly recovered in the gWAT of DIO mice administered with 10 mg/kg PDE5i compared to that of control DIO mice (Fig. 2C). Moreover, the levels of total and phosphorylated forms of eukaryotic translation initiation factor 4EBP1 and S6 phosphorylation were increased in response to insulin in gWATs of HFD-fed mice compared to those in CD-fed mice (Fig. 2C). However, PDE5i administration showed an additional increasing trend in both phosphorylated and total forms of 4EBP1, whereas S6 phosphorylation exhibited no difference (Fig. 2C). These results suggest that chronic PDE5i administration improves systemic insulin sensitivity by regulating insulin sensitivity in gWATs.
PDE5i alters the ATM polarization and inflammation in gWAT
We investigated the mechanisms underlying the improvement of insulin sensitivity in gWAT. Adipose tissue inflammation is a hallmark of insulin resistance, with ATMs playing a central role [31,34,35]. In obesity, increased M1 ATM levels and decreased M2 ATM levels are closely associated with insulin resistance [34,35]. To determine whether the PDE5i-induced improvement in insulin sensitivity is attributable to changes in ATM polarization, we conducted a FACS analysis. CD11b+F4/80+ Ly-6G− ATMs were characterized by the expression of CD11c and CD206 (Fig. 3A) [7-9]. In gWAT of HFD-fed mice, CD11c− CD206+ M2-like ATMs were significantly decreased compared to those in CD-fed mice (Fig. 3B). CD11c+ CD206− M1-like ATMs exhibited an increasing, albeit non-significant, trend in HFD-fed mice (Fig. 3B). Chronic administration of 10 mg/kg PDE5i increased the proportion of CD11c− CD206+ M2-like ATMs in the gWAT of HFD-fed mice (Fig. 3B). Consequently, PDE5i administration significantly decreased the elevated ATM M1/M2 ratio induced by HFD, reaching levels comparable to those of the CD group (Fig. 2B). These results indicated that chronic PDE5i administration may improve insulin sensitivity by reducing the M1/M2 ratio of ATMs in gWAT.
Phosphodiesterase type 5 (PDE5) inhibitor (PDE5i) administration mitigates gonadal white adipose tissue (gWAT) inflammation by modulating the immune cell population. (A) Flow cytometry gating strategies used to define CD11b+ F4/80+ CD11c+ CD206− M1-like adipose tissue macrophages (ATMs), CD11b+ F4/80+ CD11c− CD206+ M2-like ATMs, CD4+ T-cell receptor β (TCRβ)+ interferon-γ (IFNγ)+ T helper 1 (Th1) cells, and CD4+ TCRβ+ CD25+ forkhead box P3 (FoxP3)+ regulatory T (Treg) cells in gWAT. (B) Flow cytometry analysis of CD11b+ F4/80+ CD11c+ CD206− M1-like ATMs and CD11b+ F4/80+ CD11c− CD206+ M2-like ATMs in gWAT (n=5 for chow diet [CD]-vehicle group, n=4 for high-fat diet [HFD]-vehicle group, and n=6 for HFD-PDE5i group). (C) Flow cytometry analysis of CD4+ TCRβ+ IFNγ+ Th1 cells and CD4+ TCRβ+ CD25+ FoxP3+ Treg cells in gWAT (n=5 for CD-vehicle group, n=4 for HFD-vehicle group, and n=6 for HFD-PDE5i group). (D) Comparison of the mRNA expression of various cytokines (interleukin 1β [Il1β], Il6, CXC motif chemokine ligand 15 [Cxcl15], tumor necrosis factor [Tnf], Il4, Il10, and transforming growth factor β1 [Tgfβ1]) in the gWAT of mice-fed CD or HFD and treated with vehicle or PDE5i (n=5 for CD-vehicle group, n=4 for HFD-vehicle group, and n=6 for HFD-PDE5i group). Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided one-way analysis of variance (ANOVA) (B, C, D). SSC-A, side scatter area; FSC-A, forward scatter area; FSC-W, forward scatter width; FSC-H, forward scatter height; NS, non-significant. aP<0.05, bP<0.01, cP<0.001 between indicated groups.
As the activity of CD4+ T-cells can influence macrophage polarization [36,37], we examined the effects of PDE5i on CD4+ T-cell population. CD4 T-cells in gWAT were gated by TCRβ and CD4, followed by IFNγ+ for Th1 cells or CD25+ and FoxP3+ for Treg cells (Fig. 3A). CD25+ FoxP3+ Treg cells exhibited a decreasing tendency following HFD exposure; however, they were significantly increased with chronic PDE5i administration (Fig. 3C). The proportion of IFNγ+ Th1 cells did not differ among the groups (Fig. 3C). Moreover, a 7-day PDE5i administration increased the proportions of M2-like ATMs and Treg cells without affecting those of M1-like ATMs and Th1 cells (Supplementary Fig. 2D and E). These results indicate that PDE5i exhibits an immunoregulatory effect on CD4 T-cells and ATMs, thereby enhancing insulin sensitivity.
We investigated whether PDE5i administration alters the mRNA expression of inflammatory cytokines in the gWAT of HFD-fed mice. HFD increased the mRNA expression levels of proinflammatory cytokines, including Il1β, Il6, CXC motif chemokine ligand 15 (Cxcl15), and Tnf, in gWAT (Fig. 3D). However, PDE5i administration downregulated the expression of Il1β, Il6, and Tnf, while upregulating that of anti-inflammatory cytokine Il4 (Fig. 3D). These findings indicate that in addition to influencing ATM polarization, PDE5i exhibits anti-inflammatory effects in the gWAT of HFD-fed mice.
We also analyzed the effects of PDE5i on other adipose tissues, including iWAT and BAT. HFD did not significantly alter the percentages of CD11c+ CD206− M1-like ATMs or CD11c− CD206+ M2-like ATMs in the iWAT, whereas it significantly decreased M1-like ATMs and increased M2-like ATMs in the iWAT of HFD-fed mice (Supplementary Fig. 4A). Neither HFD nor PDE5i affected ATM polarization in the BAT (Supplementary Fig. 4C), and the polarization of CD4 T-cells in iWAT and BAT remained unchanged, indicating that PDE5i may more rapidly modulate the polarization of ATMs than that of CD4 T-cells (Supplementary Fig. 4B and D). The results suggest that the effects of PDE5i may vary depending on the tissue type.
Chronic PDE5i administration attenuates blood leakage in the gWAT of HFD-fed obese mice
Enhanced vascular intensity and blood perfusion in gWAT improve systemic insulin sensitivity [23]. To investigate whether PDE5i increases the vascular intensity in the gWAT of DIO mice, we examined the vascular intensities of gWATs in the CD, HFD, and HFD+PDE5i groups (Fig. 4A). Whole mount staining of CD31, a marker for blood vessels, revealed that vascular intensity was significantly decreased by HFD, and this effect was not reversed by PDE5i administration (Fig. 4A). Moreover, neither HFD feeding nor additional PDE5i administration could significantly altered the mRNA expression of endothelial cell-associated genes, such as vascular endothelial growth factor-A (Vegfa), vascular endothelial growth factor receptor 1 (Vegfr1), Vegfr2, angiopoietin-1 (Angpt1), and Angpt2; only vascular endothelial growth factor B (Vegfb) expression was increased by HFD (Supplementary Table 1, Supplementary Fig. 5).
Phosphodiesterase type 5 (PDE5) inhibitor (PDE5i) administration improves vascular perfusion in the gonadal white adipose tissue (gWAT) of high-fat diet (HFD)-fed mice. (A) Representative images and quantification of CD31+ blood vessels in gWAT (n=4). Scale bars: 200 μm. (B) Representative images and quantification of double immunostaining of CD31 and neuronglial antigen 2 (NG2) in gWAT (n=4). Scale bars: 10 μm. (C) Representative images and quantification of double immunostaining of CD31 and vascular endothelial-cadherin (VECad) in gWAT (n=4). Scale bars: 10 μm. (D) Fluorescence live images taken after tail vein injection of Evans blue dye and quantification in chow diet (CD)- or HFD-fed mice, administered with either vehicle or PDE5i (n=3). Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided oneway analysis of variance (ANOVA) (A-C, D [upper]) and one-sided two-way ANOVA (D [lower]). A.U., arbitrary unit; NS, nonsignificant. aP<0.05, bP<0.01, cP<0.001 between indicated groups; dP<0.05 indicates HFD+vehicle group vs. HFD+PDE5i group.
We subsequently examined the changes in vascular integrity in the gWAT of CD, HFD, and HFD+PDE5i groups. We performed co-immunostaining of CD31 with VECad, an adherent junction marker, or NG2, a pericyte marker. The expression of VECad and NG2 was decreased in CD31+ blood vessels in the gWAT of HFD-fed obese mice (Fig. 4B and C). However, PDE5i administration significantly increased VECad expression in the vessels, whereas NG2+ pericyte coverage remained unaffected (Fig. 4B and C). We next conducted in vivo fluorescence live imaging to assess functional blood flow. In the gWAT of HFD-fed mice, we observed rapid fluorescence leakage into the gWAT tissue compared to CD-fed mice; however, the leakage was significantly suppressed in mice administered with PDE5i (Fig. 4D). These findings suggest that PDE5i improved the blood flow in the gWAT of HFD-fed mice.
PDE5i exhibits anti-inflammatory properties in RAW264.7 cells and BMDMs
We investigated whether PDE5i directly affects gWAT ATMs to exert its anti-inflammatory effects. We activated RAW264.7 cells, a macrophage cell line, by treating them with LPS (10 ng/mL), which significantly increased Tnf expression after 2 hours, Il1β expression after 4 hours, and Il6 expression after 6 hours (Supplementary Fig. 6A). To examine the direct immunoregulatory effect of PDE5i on macrophages, LPS-stimulated RAW264.7 cells were co-treated with various PDE5i doses. PDE5i suppressed LPS-induced Tnf, Il1β, and Il6 expression in RAW264.7 cells in a dose-dependent manner (Supplementary Fig. 6B).
We further investigated the anti-inflammatory effects of PDE5i using BMDMs (Fig. 5A). Successful BMDM differentiation was confirmed using FACS analysis (Fig. 5B). We treated BMDMs with LPS as well as various doses of PDE5i, and assessed proinflammatory cytokine production via CBA and FACS (Fig. 5C). We observed that LPS significantly increased the production of IL-1α, IL-1β, IL-6, TNF-α, and monocyte chemoattractant protein 1 (MCP1), which was dose-dependently decreased by PDE5i treatment (Fig. 5C). These results demonstrate the role of PDE5i in mitigating inflammation.
Phosphodiesterase type 5 (PDE5) inhibitor (PDE5i) exhibits anti-inflammatory effects and induced M2 polarization in bone marrow-derived macrophages (BMDMs). (A) An illustration of the experimental overview to obtain BMDMs. (B) Confirmation of successful differentiation into BMDMs. (C) Flow cytometry images and quantification of cytokine production in BMDMs treated with lipopolysaccharide (LPS) or PDE5i (n=4). The concentrations of PDE5i are 25, 50, and 100 μM. (D) mRNA expression of cytokines associated with M2 polarization, including Cd36, mannose receptor C-type 1 (Mrc1), transforming growth factor β1 (Tgfβ1), and vascular endothelial growth factor-A (Vegfa) in the BMDMs (n=3). (E) Confirmation of successful shRNA transfection. (F) Flow cytometry images and quantification of cytokine production (n=4). Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided one-way analysis of variance (ANOVA) (C), one-sided two-way ANOVA (F), or two-sided unpaired Student’s t-test (D). IMDM, Iscove’s Modified Dulbecco’s Medium; M-CSF, macrophage colony-stimulating factor; BM, bone marrow; SSC-A, side scatter area; FSC-A, forward scatter area; NS, non-significant; MHC-II, major histocompatibility complex II; IL, interleukin; GFP, green fluorescent protein; TNF-α, tumor necrosis factor-α; MCP1, monocyte chemoattractant protein 1. aP<0.05, bP<0.01, cP<0.001 vs. control.
We examined whether PDE5i exerts an immunoregulatory role by directly inducing M2 polarization in BMDMs. We administered PDE5i in the presence or absence of IL-4, and evaluated the mRNA expressions of genes related to M2 polarization, including Cd36, mannose receptor C-type 1 (Mrc1), transforming growth factor β1 (Tgfβ1), and Vegfa. Under normal conditions, PDE5i treatment did not alter the expression of M2 polarization markers in BMDMs (Fig. 5D). However, in the presence of IL-4, PDE5i upragulated the mRNA expression of Mrc1, and Vegfa, indicating that it directly induced BMDMs to exhibit M2 polarization (Fig. 5D). Overall, PDE5i not only attenuates macrophage-mediated inflammation but also promotes M2 polarization, exerting immunoregulatory effects.
We investigated whether Pde5 depletion could attenuate LPS-induced inflammation by transfecting BMDMs with Pde5a shRNA and subsequently assessing the production of LPS-induced cytokines using a CBA. Successful transfection of Pde5a shRNA was confirmed via FACS (Fig. 5E). Consistent with the results observed following PDE5i treatment, Pde5 depletion in BMDMs significantly inhibited LPS-induced proinflammatory cytokine production, including that of IL-1α, IL-1β, TNF-α, and MCP-1 (Fig. 5F). Collectively, these results indicate that PDE5 plays a critical role in the inflammatory response of macrophages.
Inhibiting STAT6 signaling prevents PDE5i-induced M2 polarization and improves insulin sensitivity
We investigated the molecular mechanisms by which PDE5i exerts IL-4-induced anti-inflammatory effects, thereby improving insulin sensitivity in vivo. STAT6 is important for M2 polarization in response to IL-4 [38,39]. Therefore, we administered AS-1517499, a STAT6 phosphorylation inhibitor, to HFD-fed obese mice (Fig. 6A) as previously reported [40,41]. We observed no significant differences in body weight with either PDE5i or AS-1517499 administration (Fig. 6B). However, insulin tolerance test revealed that the PDE5i-induced improvement in insulin sensitivity was partially attenuated by AS-1517499 (Fig. 6C). Moreover, AS-1517499 significantly inhibited the increased CD11c− CD206+ M2-like polarization induced by chronic PDE5i administration, while M1 polarization was unaltered (Fig. 6D). These findings suggested that PDE5i enhances M2 polarization and improves insulin sensitivity, at least in part, through the STAT6 pathway.
Inhibiting signal transducer and activator of transcription 6 (STAT6) signaling prevents phosphodiesterase type 5 (PDE5) inhibitor (PDE5i)-induced M2 polarization and improved insulin sensitivity. (A) Experimental timeline of PDE5i or AS-1517499 administration. (B) Changes in body weight following PDE5i or AS-1517499 administration (n=5). (C) Blood glucose levels measured using insulin tolerance tests following PDE5i or AS-1517499 administration (n=5). (D) Flow cytometry analysis of CD11b+ F4/80+ CD11c+ CD206− M1-like adipose tissue macrophages (ATMs) and CD11b+ F4/80+ CD11c− CD206+ M2-like ATMs in gonadal white adipose tissue (n=5). Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided two-way analysis of variance (ANOVA) (B, C) or one-sided one-way ANOVA (D). ITT, insulin tolerance test; HFD, high-fat diet; IPITT, intraperitoneal insulin tolerance test; NS, non-significant. aP<0.05, bP<0.001, cP<0.0001 vs. control; dP<0.05, eP<0.0001 indicate HFD+PDE5i group vs. HFD+PDE5i+AS-1517499 group.
DISCUSSION
We demonstrated that chronic PDE5i administration improved insulin sensitivity, exhibited immunoregulatory effects by lowering the M1/M2 ratio, and improved blood perfusion in the gWAT of HFD-fed obese mice. Our in vitro experiments using RAW264.7 cells and BMDMs revealed that PDE5i ameliorated LPS-induced inflammation and stimulated IL-4-induced M2 polarization. These findings highlight PDE5i as a promising therapeutic target for treating inflammatory and metabolic disorders.
Adipose tissue inflammation during HFD feeding induces insulin resistance, in which ATMs play a critical role. Particularly, the reduction in M1/M2 polarization is closely associated with improved insulin resistance [42,43]. In our study, chronic PDE5i administration increased the population of CD11c− CD206+ M2-like ATMs, and decreased the M1/M2 ratio, and improved insulin sensitivity. Although various studies suggest that a decrease in the ATM M1/M2 ratio positively impacts insulin sensitivity, a consistent reduction in M1 polarization and increased M2 polarization is not always observed. Consistent with our findings, where PDE5i administration only increased M2-like polarization without a corresponding decrease in M1-like polarization, Fujisaka et al. [44] reported that IL-10 overexpression increased M2-associated mRNA expression, including that Ym1, Mrc1, and Cd163, whereas M1-associated mRNA expression, such as that of Cd11c and Tnfα exhibited no significant differences or a slight increasing trend. However, Poursharifi et al. [45] demonstrated that global depletion of α/β-hydrolase domain-containing 6 exerted anti-inflammatory effects, decreasing CD11c+ M1-like polarization without affecting CD301+ M2-like polarization. Therefore, the phenomena observed in vivo models are difficult to predict and are influenced by various factors, warranting additional research for a more comprehensive investigation.
In addition, Nawaz et al. [9] reported that depletion of CD206+ M2-like ATMs improves insulin sensitivity and significantly increases glucose and insulin metabolism under both CD and HFD conditions. They demonstrated that these ATMs induce adipocyte hypertrophy by inhibiting the differentiation and proliferation of adipocyte progenitors, leading to insulin resistance [9]. However, their study was limited, as depletion of CD206+ cells may also affect the CD11c+ CD206+ ATM population, which impairs insulin action in obesity [46]. In our FACS analysis, CD11c+ CD206+ ATMs were unaltered by PDE5i administration, although CD11c− CD206v M2-like ATM proportions were reduced in the gWAT of DIO mice (Supplementary Fig. 7). These conflicting results warrant further studies focusing on the spatiotemporal role of CD206+ ATMs in insulin resistance.
In this study, we performed a short-term (7 days) PDE5i administration to analyze the causal relationship between immune cell polarization and insulin sensitivity. Notably, ATM M2 polarization was significantly increased in the gWAT of mice administered with 10 mg/kg PDE5i. However, only a trend toward improved insulin sensitivity was observed suggesting the presence of a time gap between ATM polarization and insulin sensitivity in gWAT. This may be explained by the fact that metabolic homeostasis evolves more slowly than immune system alterations [3,47].
In addition to analyzing gWAT, we analyzed iWAT and BAT to further investigate the relationship between ATM polarization in adipose tissue and insulin sensitivity. PDE5i significantly reduced CD11c+ CD206− M1-like ATM proportions and increased CD11c− CD206+ M2-like ATM proportions in iWAT, with no significant changes observed in BAT. These results indicated that ATM polarization may vary across different adipose tissues. Notably, we have previously demonstrated that insulin sensitivity is dominantly regulated by gWAT, rather than iWAT or BAT, whereas iWAT and BAT are more closely associated with energy metabolism processes such as thermogenesis and fatty acid catabolism [31]. These findings indicate that insulin sensitivity is more pronounced in gWAT than in iWAT or BAT [31]. Moreover, BAT exhibits elevated expression of sympathetic markers, such as tyrosine hydroxylase, and fatty acid oxidation markers, including carnitine palmitoyltransferase 1, whereas gWAT demonstrates increased levels of adiponectin, a critical regulator of systemic insulin sensitivity [48-50]. These findings suggest that adipose tissues may play distinct roles in contributing to energy metabolism and insulin sensitivity.
The mTOR signaling pathway is closely associated with insulin signaling [33]. In our study, we found that HFD increased the levels of 4EBP1 and the S6 phosphorylation in gWAT. Moreover, chronic PDE5i administration showed an additional increasing trend in 4EBP1 levels. This result suggests that the improved insulin sensitivity induced by PDE5i may be linked to the upregulation of 4EBP1. Consistently, a previous study using 4Ebp1 transgenic mice demonstrated that 4Ebp1 overexpression significantly improved insulin sensitivity in HFD-fed C57BL/6J male mice [51]. A study using mice with adipose tissue-specific deletion of liver kinase B1 (LKB1) and mTOR demonstrated severe insulin resistance without affecting body weight or glucose metabolism, aligning with the phenotypes observed in our mouse model [52]. However, mTOR signaling activation under HFD conditions can elicit variable results depending on the experimental conditions. Unlike our observation where HFD-fed mice exhibited increased 4EBP1 expression and S6 phosphorylation in the gWAT, another study reported a reduction in 4EBP1 levels in visceral fat [51]. These discrepancies might be attributed to differences in mTOR signaling depending on the HFD exposure period or the presence or absence of insulin administration. Therefore, in-depth studies exploring the interactions among mTOR signaling, PDE5i, and immune cells are essential to better understand the underlying mechanisms.
Recently, a positive correlation has been reported between adipose tissue vascular intensity and insulin sensitivity [23]. Specifically, Vegfr1 depletion enhanced the VEGFR2 angiogenic signal, which increases vascular intensity in the adipose tissue, resulting in improved glucose and insulin sensitivity [23]. Consistently, we observed that chronic PDE5i administration not only recovered VECad expression but also improved blood perfusion in gWAT. These results indicate a positive correlation between improved blood flow in gWAT and enhanced insulin sensitivity although a complete restoration of pericyte coverage was not observed. Given that PDE5i exerts anti-inflammatory effects on ATMs, the normalized vascular VECad expression and enhanced blood flow may be due to reduced inflammation. Therefore, further investigations of the vascular-immune cell interactions in gWAT through cell-specific loss-of-function studies are needed.
In summary, our study underscores the potential therapeutic value of PDE5i in improving insulin sensitivity by regulating adipose tissue immune cells and improving blood flow in gWAT under HFD conditions.
SUPPLEMENTARY MATERIALS
Supplementary materials related to this article can be found online at https://doi.org/10.4093/dmj.2024.0308.
Primer sequences used for quantitative polymerase chain reaction analysis
High-fat diet (HFD) induces obesity and insulin resistance. (A) Changes in body weight during HFD (n=10 for chow diet [CD] and n=6 for HFD). (B) Blood glucose level measured by insulin tolerance test, performed after obesity induction (n=10 for CD and n=6 for HFD). Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided two-way analysis of variance (ANOVA). aP<0.001 between indicated groups.
Short-term phosphodiesterase type 5 (PDE5) inhibitor (PDE5i) administration slightly improved insulin sensitivity and significantly enhanced M2-like polarization in high-fat diet (HFD) mice. (A, B) Changes in body weight and food intake after PDE5i short-term administration (n=7 for control, n=6 for PDE5i 5 mg/kg, and n=7 for PDE5i 10 mg/kg). (C) Insulin tolerance tests were performed 1 week after vehicle or PDE5i administration (n=5). (D) Flow cytometry analysis of the CD11b+ F4/80+ CD11c+ CD206− M1-like adipose tissue macrophages (ATMs) and CD11b+ F4/80+ CD11c− CD206+ M2-like ATMs in gonadal white adipose tissue (gWAT) (n=5). (E) Flow cytometry analysis of the CD4+ T-cell receptor β (TCRβ)+ interferon-γ (IFNγ)+ T helper 1 (Th1) cells and CD4+ TCRβ+ CD25+ forkhead box P3 (FoxP3)+ regulatory T (Treg) cells in gWAT (n=5). Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided two-way analysis of variance (ANOVA) (A-C) or one-sided one-way ANOVA (D, E). IPITT, intraperitoneal insulin tolerance test; NS, not significant. aP<0.05, bP<0.01 between indicated groups.
Glucose tolerance is unchanged by chronic phosphodiesterase type 5 (PDE5) inhibitor (PDE5i) administration. Glucose tolerance tests were performed after vehicle or PDE5i administration (n=7 for control, n=6 for PDE5i 5 mg/kg, and n=7 for PDE5i 10 mg/kg). Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided two-way analysis of variance (ANOVA). HFD, high-fat diet; OGTT, oral glucose tolerance test.
Phosphodiesterase type 5 (PDE5) inhibitor (PDE5i) significantly reduced M1-like adipose tissue macrophages (ATMs) and increased M2-like ATMs in inguinal white adipose tissue (iWAT) of high-fat diet (HFD)-fed mice. (A) Flow cytometry analysis of the CD11b+ F4/80+ CD11c+ CD206− M1-like ATMs and CD11b+ F4/80+ CD11c− CD206+ M2-like ATMs in iWAT (n=4). (B) Flow cytometry analysis of the CD4+ T-cell receptor β (TCRβ)+ interferon-γ (IFNγ)+ T helper 1 (Th1) cells and CD4+ TCRβ+ CD25+ forkhead box P3 (FoxP3)+ regulatory T (Treg) cells in iWAT (n=4). (C) Flow cytometry analysis of the CD11b+ F4/80+ CD11c+ CD206− M1-like ATMs and CD11b+ F4/80+ CD11c− CD206+ M2-like ATMs in brown adipose tissue (BAT) (n=4). (D) Flow cytometry analysis of the CD4+ TCRβ+ IFNγ+ Th1 cells and CD4+ TCRβ+ CD25+ FoxP3+ Treg cells in BAT (n=4). Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided one-way analysis of variance (ANOVA). CD, chow diet; NS, not significant. aP<0.01, bP<0.001 between indicated groups.
mRNA expressions related to vascular-related genes are unchanged by high-fat diet (HFD) or phosphodiesterase type 5 (PDE5) inhibitor (PDE5i) administration. (A-F) Comparison of the mRNA expression of vascular-related genes, including vascular endothelial growth factor-A (Vegfa), vascular endothelial growth factor B (Vegfb), vascular endothelial growth factor receptor 1 (Vegfr1), Vegfr2, angiopoietin-1, and angiopoietin-2 in gonadal white adipose tissue (gWAT) (n=5 for control, n=6 for PDE5i 5 mg/kg, and n=6 for PDE5i 10 mg/kg). Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided one-way analysis of variance (ANOVA). CD, chow diet. aP<0.05 between indicated groups.
Phosphodiesterase type 5 (PDE5) inhibitor (PDE5i) prevents lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. (A) The changes in tumor necrosis factor (Tnf), interleukin 1β (Il1β), and Il6 expressions in RAW264.7 cells following LPS treatment (n=6). (B) Changes in the expression of inflammatory cytokines including Tnf, Il1β, and Il6 in RAW264.7 cells after LPS treatment, depending on the concentration of PDE5i. The expression levels of Tnf, Il1β, and Il6 were measured at 6 hours following LPS treatment. The concentrations of PDE5i are 1, 10, 50, and 100 μM. Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided one-way analysis of variance (ANOVA). aP<0.05, bP<0.001 vs. control.
Percentage of CD11c+ CD206+ double positive adipose tissue macrophages (ATMs) are unchanged by high-fat diet (HFD) or chronic phosphodiesterase type 5 (PDE5) inhibitor (PDE5i) administration. Flow cytometry analysis of CD11b+ F4/80+ CD11c+ CD206+ ATMs in gonadal white adipose tissue (n=5 for CD-vehicle, n=4 for HFD-vehicle, and n=6 for HFD-PDE5i). Results are presented as mean±standard error of the mean. Statistical analyses were performed using one-sided one-way analysis of variance (ANOVA). CD, chow diet.
Notes
CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was reported.
AUTHOR CONTRIBUTIONS
Conception or design: J.H.S., C.H.L.
Acquisition, analysis, or interpretation of data: all authors.
Drafting the work or revising: J.H.S., C.H.L.
Final approval of the manuscript: all authors.
FUNDING
This work was supported by the National Research Foundation of Korea (NRF) grant (2022R1C1C1004187 and RS-2023-00223501; Bio&Medical Technology Development Program) funded by the Korea government (MSIT) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) (HR21C0198) funded by the Ministry of Health & Welfare.
ACKNOWLEDGMENTS
We used ChatGPT-3.5 to check the grammar of some sentences.