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Rationale: The diabetes mellitus drug metformin is under investigation in cardiovascular disease, but the molecular mechanisms underlying possible benefits are poorly understood.
Objective: Here, we have studied anti-inflammatory effects of the drug and their relationship to antihyperglycemic properties.
Methods and Results: In primary hepatocytes from healthy animals, metformin and the IKKβ (inhibitor of kappa B kinase) inhibitor BI605906 both inhibited tumor necrosis factor-α–dependent IκB degradation and expression of proinflammatory mediators interleukin-6, interleukin-1β, and CXCL1/2 (C-X-C motif ligand 1/2). Metformin suppressed IKKα/β activation, an effect that could be separated from some metabolic actions, in that BI605906 did not mimic effects of metformin on lipogenic gene expression, glucose production, and AMP-activated protein kinase activation. Equally AMP-activated protein kinase was not required either for mitochondrial suppression of IκB degradation. Consistent with discrete anti-inflammatory actions, in macrophages, metformin specifically blunted secretion of proinflammatory cytokines, without inhibiting M1/M2 differentiation or activation. In a large treatment naive diabetes mellitus population cohort, we observed differences in the systemic inflammation marker, neutrophil to lymphocyte ratio, after incident treatment with either metformin or sulfonylurea monotherapy. Compared with sulfonylurea exposure, metformin reduced the mean log-transformed neutrophil to lymphocyte ratio after 8 to 16 months by 0.09 U (95% confidence interval, 0.02–0.17; P=0.013) and increased the likelihood that neutrophil to lymphocyte ratio would be lower than baseline after 8 to 16 months (odds ratio, 1.83; 95% confidence interval, 1.22–2.75; P=0.00364). Following up these findings in a double-blind placebo controlled trial in nondiabetic heart failure (trial registration: NCT00473876), metformin suppressed plasma cytokines including the aging-associated cytokine CCL11 (C-C motif chemokine ligand 11).
Conclusion: We conclude that anti-inflammatory properties of metformin are exerted irrespective of diabetes mellitus status. This may accelerate investigation of drug utility in nondiabetic cardiovascular disease groups.
Clinical Trial Registration: Name of the trial registry: TAYSIDE trial (Metformin in Insulin Resistant Left Ventricular [LV] Dysfunction). URL: https://www.clinicaltrials.gov. Unique identifier: NCT00473876.
Metformin is the first-line drug in type 2 diabetes mellitus because compared with other type 2 diabetes mellitus treatments, in both clinical trials and in observational studies, metformin monotherapy is associated with fewer adverse cardiovascular events,1,2 and in some studies, a reduced risk of cancer.3 The reasons for this relative benefit are unclear, and metformin’s molecular action is a vigorous area of current research.4–7 Metformin’s chemical properties include a strongly hydrophilic character, metal-binding properties, and a pKa within the physiological pH range.6–8 The key clinical hallmark of metformin’s antihyperglycemic action is suppression of hepatocyte gluconeogenesis.4,5,9 The most likely cellular effect underlying this response is inhibition of mitochondrial enzymes, including complex I in the electron transport chain.10,11 More recently, mitochondrial glycerophosphate dehydrogenase has been suggested as an alternative target.12 Mitochondrial inhibition activates AMP-activated protein kinase (AMPK),13 and recent work suggests that duodenal AMPK contributes toward effects of the drug on hepatic glucose production.14 Other studies indicate that metformin also suppresses glucose production by AMPK-independent mechanisms,12,15,16 but more broadly, AMPK may still contribute to metformin-dependent regulation of other aspects of metabolic control, such as lipogenic gene expression.4
The mechanism(s) underlying metformin’s advantage in incidence of cardiovascular disease (CVD) are unlikely to depend on effects of the drug on hyperglycemia, which is controlled equally well by metformin and insulin secretagogues.2 In addition, in animals, metformin suppresses infarct size and adverse remodeling in diabetic and nondiabetic rodents17–21 and retards heart failure progression in nondiabetic dogs.22 A better understanding of such glucose-independent properties might foster a more rational, less empirical exploitation of metformin in nondiabetic CVD. Inflammation, including nuclear factor-κB (NF-κB) signaling, is increasingly recognized as a significant contributing factor to diabetes mellitus (DM) and CVD,23,24 and several previous studies have found that metformin inhibits NF-κB signaling, including in vascular tissue25 and recently in hepatocytes.26 In the current study, we have used multiple approaches, including human studies, to define anti-inflammatory actions of metformin that may be separated from its antihyperglycemic action.
Animal and Cell Studies
Metformin and rapamycin came from calbiochem, 5-aminoimidazole-4-carboxamide riboside (AICAR) and A769662 (Tocris), tumor necrosis factor-α (TNF-α) (e-bioscience), recombinant CINC1/chemokine (C-X-C motif) ligand (CXCL) 1, C-C motif chemokine ligand (CCL)-11, interleukin (IL)-2, IL-4, stromal cell–derived factor and CCL22 (R&D systems), mouse IL-6 (Sigma), and recombinant mouse IL-1β (Life Technologies). The phospho–acetyl-CoA carboxylase Ser79 antibody was a generous gift from the DSTT (University of Dundee). The total acetyl-CoA carboxylase (Cat. number 3662), total AMPKα (2603), phospho-AMPKα Thr172 (2535), total S6 (2217), phospho-S6 Ser240/244 (2215), total p70 S6 kinase (2708), phospho-p70 S6 kinase Thr389 (9205), phospho-Raptor Ser 792 (2083), phospho inhibitor of kappa B kinase (IKK) α/β Ser176/177 (2078), IKKα/β Ser176/180 (2697), total IκB, pNF-κB, total IKKα, and total IKKβ (NF-κB sampler kit 9936) antibodies were from CST. Antisheep horseradish peroxidase (31480) and antirabbit horseradish peroxidase (31460) both came from Thermo and antimouse horseradish peroxidase was from Calbiochem (JA1200). BI605906 was generously gifted by Prof Sir Philip Cohen (Dundee).
C57BL/6 female mice (Charles River; 8–41 weeks) were maintained under a 12 hours:12 hours light:dark cycle (holding room lights on at 06:00 and off at 18:00) at 22±1°C and 50% humidity. Mice had ad libitum access to standard chow diet (7.5% fat, 75% carbohydrate, and 17.5% protein by energy [RM1 diet; Special Diet Services]) and water. All animal care protocols and procedures were performed in accordance with current regulations.
Cell Culture and Lysis for Immunoblotting
All cells were grown in an incubator at 37°C and 5% CO2. Primary mouse hepatocytes were extracted and maintained essentially as described previously.6,15
Bone marrow–derived macrophages (BMDMs) were grown from mouse bone marrow in RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies) and 10-ng/mL macrophage colony-stimulating factor (R&D systems). Cells were given fresh medium and growth factor on day 3 of culture. On day 6, BMDM cultures were supplemented with 100-ng/mL interferon γ (for M1 differentiation; R&D systems), 20-ng/mL IL-4 (for M2 differentiation; R&D systems), or 100-ng/mL lipopolysaccharide (for activation; premium grade from Sigma, expected to activate toll-like receptor [TLR]-2 and TLR4) in the presence or absence of drug treatments for the final 24 hours.
Before SDS-PAGE, cells were lysed by scraping into buffer A (50 mmol/L Tris acetate pH 7.5, 1% (wt/vol) Triton X100, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.27 mol/L sucrose, 50 mmol/L NaF, 1 mmol/L sodium orthovanadate, 10 mmol/L β-glycerophosphate, 5 mmol/L sodium pyrophosphate, 1 mmol/L benzamidine, 0.2 mmol/L phenylmethylsulfonyl fluoride, and 0.1% (v/v) β-mercaptoethanol) and then prepared for SDS-PAGE as described in the previous work.6 Immunoblot densitometry for each antibody was performed with Image Studio Lite version 5.2 (LI-COR). Each blot is representative of experiments preformed at least 3×.
Treatment of cells for hepatocyte glucose production was performed essentially as described previously, using primary mouse hepatocytes plated in 12-well plates (1.25×105 cells per well).6,15,27 Glucose production was determined after a 12-hour incubation period in glucose-free DMEM (11966; Life Technologies) supplemented with 1% pen/strep, lactate (Sigma)/pyruvate (Life Technologies; 10:1 mmol/L), and 100 nmol/L dexamethasone (dex; Merck) with or without drugs/cytokines under investigation. At the end of the incubation period of 12 hours, 500 μl of medium was collected and glucose concentration determined by GAGO assay (glucose [glucose oxidase]; Sigma) by a modified protocol scaled down to a 96-well plate format. Each column consists of data from at least 12 wells of cells, 6 each from 2 mice.
Real-Time-Polymerase Chain Reaction
Total RNA from primary mouse hepatocytes was extracted using QIAshredder (Qiagen) and Rneasy MINI KIT (Qiagen). cDNA was synthesized using RQ1 Rnase-Free Dnase kit (Promega) and ImProm-II Reverse Transcription System (Promega). Nucleospin RNA II Total RNA isolation kit (Macherey-Nagel) was used to isolate RNA from macrophages. cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (4368814, Thermo Fisher Scientific). Real-time polymerase chain reaction was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan 2× Universal PCR Master Mix (Applied Biosystems) and primer/probes mixes as stated (Applied Biosystems). Primer sets used were as follows: IL-6 Mm00446190_m1, CXCL1 Mm04207460_m1, 18S Hs03003631_g1, IL-1β Mm00434228_m1, CXCL2 Mm00436450_m1, peroxisome proliferator–activated receptor-γ m01184322_m1, fatty acid synthase Mm00662319_m1, CCL22 Mm00436439_ml, CXCL12 Mm00445553_ml, TATA-binding protein Mm01277042_m1, and sterol regulatory element-binding protein 1c Mm00550338_m1. Cycling conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 s and 60°C for 1 minute. Expression is expressed relative to 18s mRNA for hepatocytes and TATA-binding protein for macrophages (Applied Biosystems) using the 2-ΔΔCt method. Each column is composed of data from at least 3 separate experiments.
BMDMs were harvested from culture plates using 4 mmol/L EDTA in PBS for 10 minutes at 37°C. Cells were washed in flow cytometry buffer (PBS with 2% fetal bovine serum and 1 mmol/L EDTA) and stained using the following antibodies (all BD Bioscience unless stated): F4/80 (BM8; e-bioscience), CD11c (HL3), CD206 (C068C2; Biolegend), CD69 (H1.2F3), and CD40 (3/23). Fc block (4.4G2) was included in all stains. Data were acquired on a LSR II flow cytometer (Becton Dickinson) and analyzed using FlowJo software (TreeStar). BMDM culture supernatants were collected after 24-hour treatment with the differentiation or activation conditions. Levels of cytokines were quantified by standard sandwich ELISA using paired antibody kits (e-bioscience).
Validation in Clinical Patients
We validated the animal study findings in clinical patients utilizing 2 approaches: a retrospective population cohort study and a randomized placebo-controlled study of metformin. All patients provided written informed consent to participate in these clinical studies that were approved the local ethics committee.
Population Cohort Study: Metformin Exposure in DM Patients and Neutrophil to Lymphocyte Ratio.
In the population cohort study, we investigated whether the anti-inflammatory signature of metformin could be detected in humans with DM, using the GoDARTS (Genetics of Diabetes Audit and Research in Tayside Scotland) DM register.28 We compared the effect of metformin and sulfonylureas on the neutrophil to lymphocyte ratio (NLR), a marker of inflammation derived from a combination of hematological components of the systemic inflammatory response29,30 that has recently been found to be a predictor of all-cause mortality and cardiac events.31 We analyzed data from type 2 diabetes mellitus patients recruited in Tayside, Scotland, UK, between October 1, 1997, and March 1, 2010. Of the 9205 subjects with DM within the GoDARTS study, we chose 3575 treatment naive patients who were either incident metformin users or incident sulfonylurea users (but not both) and noninsulin users. Incident use meant at least 6 months before first observed metformin/sulfonylurea prescription date during which they were observable for drugs. Of these 670 patients (mean [SD]: age, 65  years; 54% men) had derived NLR values both at baseline (up to 120 days before first metformin/sulfonylurea prescription) and follow-up (8–16 months after baseline). NLR was calculated as the ratio of the neutrophil:lymphocyte count, both obtained from the same blood sample. A total of 498 (74%) patients were treated with metformin and 172 (26%) with sulfonylurea. Multivariate linear and logistic regression models were run on the 8- to 16-month follow-up NLR against the treatment group, controlling for covariates including age, sex, and baseline NLR value.
Randomized Placebo-Controlled Study: Metformin Exposure and Cytokine Levels in Nondiabetic Heart Failure Patients
The anti-inflammatory effects of metformin were investigated in a randomly selected subset of patients who had participated in a double-blind, placebo-controlled study (www.clinicaltrials.gov: NCT00473876) that had evaluated the impact of metformin on insulin resistant (IR) and exercise capacity in nondiabetic patients with congestive heart failure.32