Metformin-induced glucagon-like peptide-1 secretion contributes to the actions of metformin in type 2 diabetes (2024)

BACKGROUND. Metformin reduces plasma glucose and has been shown to increase glucagon-like peptide 1 (GLP-1) secretion. Whether this is a direct action of metformin on GLP-1 release, and whether some of the glucose-lowering effect of metformin occurs due to GLP-1 release, is unknown. The current study investigated metformin-induced GLP-1 secretion and its contribution to the overall glucose-lowering effect of metformin and underlying mechanisms in patients with type 2 diabetes.

METHODS. Twelve patients with type 2 diabetes were included in this placebo-controlled, double-blinded study. On 4 separate days, the patients received metformin (1,500 mg) or placebo suspended in a liquid meal, with subsequent i.v. infusion of the GLP-1 receptor antagonist exendin9-39 (Ex9-39) or saline. During 240 minutes, blood was sampled. The direct effect of metformin on GLP-1 secretion was tested ex vivo in human ileal and colonic tissue with and without dorsomorphin-induced inhibiting of the AMPK activity.

RESULTS. Metformin increased postprandial GLP-1 secretion compared with placebo (P = 0.014), and the postprandial glucose excursions were significantly smaller after metformin + saline compared with metformin + Ex9-39 (P = 0.004). Ex vivo metformin acutely increased GLP-1 secretion (colonic tissue, P < 0.01; ileal tissue, P < 0.05), but the effect was abolished by inhibition of AMPK activity.

CONCLUSIONS. Metformin has a direct and AMPK-dependent effect on GLP-1–secreting L cells and increases postprandial GLP-1 secretion, which seems to contribute to metformin’s glucose-lowering effect and mode of action.

TRIAL REGISTRATION. NCT02050074 (https://clinicaltrials.gov/ct2/show/NCT02050074).

FUNDING. This study received grants from the A.P. Møller Foundation, the Novo Nordisk Foundation, the Danish Medical Association research grant, the Australian Research Council, the National Health and Medical Research Council, and Pfizer Inc.

Study patients.

Twelve patients with type 2 diabetes (8 females, age 60.7 ± 8.8 years (mean ± SD); BMI, 29.8 ± 3.0 kg/m²; glycosylated hemoglobin A_1c, 6.5 ± 0.5% (47.5 ± 6.0 mmol/mol); fasting plasma glucose, 7.7 ± 1.0 mmol/l; diabetes duration, 8.1 ± 3.1 years) were included in the study. The patients were all treated with diet in combination with metformin monotherapy (n = 11) or metformin plus gliclazide (n = 1). None of the patients suffered from diabetic complications. Twenty-one patients were screened. The data (time points 0–240 minutes) from the day of placebo + Ex9-39 from 1 patient are missing, as the patient vomited twice after ingestion of the liquid meal. These data points are excluded from the figures. In order to perform the 1-way repeated measures ANOVA and post hoc Tukey’s multiple comparisons test, the missing values were extrapolated from the mean values of the other 11 patients, who completed the placebo + Ex9-39 day.

Metformin increases postprandial GLP-1 in patients with type 2 diabetes.

Time courses of GLP-1 plasma concentrations and incremental AUCs (iAUCs) are illustrated in Figure 1A, and AUCs are given in Table 1. Basal GLP-1 concentrations did not differ between the days. The GLP-1 iAUC was significantly greater after metformin + saline vs. placebo + saline (P = 0.014). There was a tendency toward Ex9-39–induced GLP-1 secretion after metformin compared with saline (P = 0.08). GLP-1 iAUC was significantly greater after metformin + Ex9-39 vs. placebo + Ex9-39 (P = 0.026). Time courses and iAUCs of glucagon plasma concentrations are illustrated in Figure 1B, and AUCs are given in Table 2. On the 2 days with Ex9-39 infusion, mean basal concentrations of plasma glucagon were higher compared with the 2 days of saline infusion (25.9 ± 2.5 nmol/l vs. 23.6 ± 2.1 nmol/l, P = 0.018). Glucagon concentrations, expressed as iAUC, were increased by metformin + Ex9-39 compared with placebo + saline (P = 0.004). Glucagon concentrations increased after meal ingestion on all days.

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Figure 1

Time courses and incremental AUCs (iAUC) of postprandial plasma concentrations of glucagon-like peptide 1 (GLP-1) and glucagon.

Time courses (0–240 minutes) and iAUCs of postprandial GLP-1 (A) and glucagon (B), after receiving a single dose of metformin (1,500 mg) or placebo with subsequent i.v. infusion of saline or exendin9-39 (Ex9-39), respectively, in 12 patients with type 2 diabetes. Data are means ± SEM. *P < 0.05, **P < 0.01. Comparisons performed by 1-way repeated measures ANOVA with Tukey’s multiple comparison post hoc test.

Table 2

Glucose, insulin, C-peptide, ISR, and glucagon

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Table 1

GLP-1, acetaminophen, total bile acids, and gallbladder volume

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Metformin reduces postprandial plasma glucose — partly via GLP-1 signaling.

Mean Ex9-39 concentrations from time 0–240 minutes were similar between the metformin + Ex-9-39 and placebo + Ex9-39 days (305 ± 15 nmol/l vs. 301 ± 17 nmol/l, P = 0.723) (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.93936DS1). Time courses of plasma glucose concentrations are illustrated in Figure 2A, and AUCs are given in Table 2. On the 2 days with Ex9-39 infusion, mean basal concentrations of plasma glucose were higher compared with the 2 days of saline infusion (9.8 ± 0.58 mmol/l vs. 8.7 ± 0.52 mmol/l, P = 0.003). Therefore, we used iAUCs to evaluate the impact of metformin and Ex9-39 on postprandial glucose tolerance. The glucose iAUC after metformin + saline was significantly smaller than the iAUC for placebo + saline (P = 0.04), whereas the difference between metformin and placebo during GLP-1 receptor blockage did not reach statistical significance (metformin + Ex9-39 vs. placebo + Ex9-39, P = 0.053). The glucose iAUC after metformin + saline was significantly smaller than the iAUC for metformin + Ex9-39 (P = 0.004). Based on individual iAUC values, the relative contribution of GLP-1 to the acute glucose-lowering effect of metformin was 75% ± 35%, calculated as follows: 100% × ([iAUC_{placebo + saline} – iAUC_{metformin + saline}] – [iAUC_{placebo + Ex9–39} – iAUC_{metformin + Ex9–39}])/(iAUC_{placebo + saline} – iAUC_{metformin + saline}) (P = 0.05). Using a 2-way ANOVA, both metformin and Ex9-39 were shown to significantly affect postprandial plasma glucose (iAUC) (P = 0.005 and P = 0.002, respectively), but no interaction between the 2 factors was evident. The time courses of the C-peptide/glucose ratios are illustrated in Figure 2B, and the AUCs for C-peptide/glucose, insulin/glucose, and insulin secretion rate (ISR)/glucose ratios are given in Table 2. Mean basal insulin/glucose ratio, C-peptide/glucose, and ISR/glucose ratio were lower on the 2 days of Ex9-39 infusion vs. the 2 days of saline infusion (8.3 pmol/mmol ± 1.2 vs. 12.4 pmol/mmol ± 1.7, 73.5 pmol/mmol ± 8.4 vs. 93.7 pmol/mmol ± 10.1, and 0.3 ± 0.0 vs. 0.2 ± 0.1, respectively). Postprandial values of insulin/glucose ratio did not differ between the interventions (Supplemental Figure 2), but C-peptide/glucose and ISR/glucose ratios were higher after metformin + saline compared with placebo + saline (P = 0.004 and P = 0.005, respectively), and placebo + Ex9-39 (P = 0.008 and P < 0.001, respectively). The ISR/glucose ratios were also higher after metformin + saline compared with metformin + Ex9-39 (P = 0.040), and the same tendency was seen regarding the effect on C-peptide/glucose ratio (P = 0.051). Indices of gastric emptying are given in Table 1. Metformin + saline significantly reduced gastric acetaminophen total AUC (tAUC_{0–60 min}) compared with metformin + Ex9-39 (P = 0.04), indicating accelerated gastric emptying induced by Ex9-39. There was no difference between metformin and placebo on either tAUC_{0–60 min} or time to maximal plasma concentration (T_max). (Supplemental Figure 3). None of the interventions affected appetite perceptions, thirst (Supplemental Figure 4), or ad libitum food intake significantly (Supplemental Figure 5).

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Figure 2

Time courses and incremental AUCs (iAUC) of postprandial plasma concentrations of glucose and the C-peptide/glucose ratio.

Time courses (0–240 minutes) and iAUCs of postprandial glucose (A) and C-peptide/glucose ratio (B), after receiving a single dose of metformin (1,500 mg) or placebo with subsequent i.v. infusion of saline or exendin9-39 (Ex9-39), respectively, in 12 patients with type 2 diabetes. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Comparisons performed by 1-way repeated measures ANOVA with Tukey’s multiple comparison post hoc test.

Metformin and Ex9-39 have no effect on postprandial plasma cholecystokinin (CCK), gallbladder motility, or total serum bile acids.

On all 4 days, CCK concentrations increased immediately after ingestion of the liquid meal, but no differences in the responses were observed (Supplemental Figure 6). Indices of gallbladder motility are given in Table 1. Baseline gallbladder volume and gallbladder contraction between 0–60 minutes were similar between the days. The refilling of the gallbladder from 60–240 minutes was similar after metformin + saline vs. placebo + saline. The gallbladder refilling was also similar after Ex9-39 with either metformin or placebo (Supplemental Figure 7). AUCs of total serum bile acids are given in Table 1. Basal concentrations of total bile acids did not differ between the 4 study days. On all 4 days after ingestion of the liquid meal, serum bile acids increased rapidly and peaked after 45–60 minutes. The iAUC for total bile acids did not differ after metformin + saline vs. placebo + saline (P = 0.87) and did not appear to be affected by Ex9-39 (Supplemental Figure 8).

Metformin triggers GLP-1 secretion in ex vivo human ileal and colonic mucosae.

To understand how metformin causes GLP-1 release, we utilized an ex vivo secretion approach using human gut epithelial tissue sections. Exposure to high external K⁺ (70 mM) or to a combination of known activators of L cell secretion forskolin (FSK, 10 μM) and 3-isobutyl-1-methylxanthine (IBMX, 10 μM) both increased intact GLP-1 release from colonic epithelial tissue (n = 22, P < 0.001, Figure 3A). Acute exposure to metformin (10 μM) also triggered an increase in intact GLP-1 levels (n = 46, P < 0.01, Figure 3B). We saw a similar increase in GLP-1 release in response to metformin in ileal tissue (n = 10, P < 0.05, Figure 3C). Thus, exposure of metformin increases GLP-1 release within 15 minutes from human colonic and ileal L cells. To identify whether the effect of metformin on L cell secretion was altered in human obesity or type 2 diabetes, we examined responses to metformin in our colonic preparation across BMI and in samples obtained from patients with type 2 diabetes. Neither the basal release of GLP-1, nor the degree of metformin-stimulated GLP-1 release, correlated with BMI (n = 46, Supplemental Figure 9). No difference was seen in either basal or stimulated GLP-1 release between tissue obtained from nondiabetes (n = 35) or type 2 diabetes individuals (n = 11, Supplemental Figure 9). Thus, basal L cell secretion, and the response of L cells to metformin, do not seem to change across BMI and appear unrelated to diabetes status. We investigated the mechanism by which metformin triggers L cell secretion. AMPK has been associated with metformin action (4), and inhibiting AMPK activity using 10 μM dorsomorphin blocked metformin-induced GLP-1 secretion (n = 20, Figure 3D). We then used a series of membrane transporter antagonists to identify the mechanism of metformin internalization in human colonic L cells. Quinine (organic cation transporter 1 [OCT1] inhibitor, 1 mM) and lopinavir (plasma membrane monoamine transporter [PMAT] inhibitor, 10 μM) had no effect on metformin-induced GLP-1 release, while fluoxetine (serotonin transporter [SERT] inhibitor, 1 μM) blocked metformin-induced GLP-1 release (n = 18, Figure 3E).

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Figure 3

Metformin-induced secretion in human L cells.

(A) Colonic epithelial preparations readily secrete glucagon-like peptide 1 (GLP-1) in response to high (70 mM) external K⁺ or to a combination of 3-isobutyl-1-methylxanthine (IBMX) and forskolin (FSK) (n = 22). (B and C) Metformin (10 μM) increases GLP-1 release after 15 minutes in epithelial tissue from human colon (n = 46) (B) and ileum (n = 10) (C). (D) Metformin-induced GLP-1 release is blocked by the AMP-activated protein kinase (AMPK) inhibitor dorsomorphin (n = 18). (E) Metformin-induced GLP-1 release is blocked by the serotonin transporter (SERT) inhibitor fluoxetine and the plasma membrane monoamine transporter (PMAT) lopinavir (n = 18). Bar graph data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with respective control groups. Comparisons performed by paired 1-way ANOVA and paired Student’s t test.

Clinical study design.

The clinical study was a randomized, double-blinded, placebo-controlled study. Screening visits and all experiments were conducted at Center for Diabetes Research at Gentofte Hospital. A flowchart is provided to report the flow of participants through the study and to indicate participants who were included or excluded from the final analysis (Figure 4). The patients were randomized to a random intervention sequence (using www.random.org) by an unblinded colleague at Center for Diabetes Research, and liquid meals and i.v. infusions (see below) were prepared by laboratory technicians. The principal investigator (MH) remained blinded for the sequence of the study procedures. Power calculations were based on minimal relevant change in postprandial plasma GLP-1 concentrations assessed as AUC. Other endpoints included change in plasma insulin, glucagon, CCK, total plasma bile acid concentrations, plasma concentrations of Ex9-39 (on the 2 days of infusion of Ex9-39), and serum acetaminophen (for evaluation of gastric emptying). Furthermore, gallbladder volume, appetite, and ad libitum lunch intake were evaluated.

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Figure 4

CONSORT diagram of the clinical study.

The flow chart shows the number of patients who were screened, allocated to treatment, completed the study, and included in the final analysis. Ex9-39, exendin9-39.

Eligibility criteria.

Prior to the experiments, patients were screened according to inclusion and exclusion criteria, including family history, physical examination, standard blood panel, and urine for albumin and creatinine. Inclusion criteria included White race, BMI > 25 kg/m², older than 18 years of age, normal hemoglobin, normal thyroid function (thyroid-stimulatory hormone within normal range), and informed consent. Exclusion criteria included a history of abdominal surgery, gastrointestinal disorders including gallstone disease, nephropathy (creatinine > 130 μmol/l and/or albuminuria), liver disease (alanine aminotransferase and/or aspartate aminotransferase ≥ 2 times normal serum upper limit) and treatment with DPP-4 inhibitors, GLP-1 receptor agonists and/or insulin, and treatment with medicine that could not be paused for 12 hours.

Experimental procedures.

The patients were studied on 4 separate experimental days separated by at least 48 hours. On all 4 days, the patients were studied in recumbent position after a 10-hour overnight fast including liquids, medication, and tobacco use. The patients were instructed not to take their antidiabetic medication for 1 week prior to each experimental day and to refrain from exercise on experiment days and the day before. After arriving at the lab, an i.v. catheter was inserted into a cubital vein in each arm (1 for blood sampling and 1 for infusion). The forearm used for blood sampling was wrapped in a heating pad (50°C) for arterialization of venous blood during the experiment. Continuous isovolemic infusions of either saline or Ex9-39 were then started at a rate of 10 ml/hour by the use of a pump (Terufusion Infusion Pump, TE-171, Terumo), for Ex9-39 corresponding to a rate of 450 pmol × kg body weight^–1 × min^–1, since this dose has previously been shown to block the insulinotropic effect of synthetic GLP-1 by ~90% (64). After 30 minutes, at time 0 minutes, the patients ingested a 200 ml, 1,264 kJ (302 kcal) mixed liquid meal (chocolate-flavored Nutridrink, 36.8 g carbohydrates, 12.0 g protein and 11.6 g fat; Nutricia) mixed with a dispersion of 1,500 mg of acetaminophen in 100 ml of water (for evaluation of gastric emptying) and metformin 1,500 mg or placebo. The dispersions were mixed for 20 minutes at room temperature before intake. The patients were instructed to ingest the 300 ml of liquid over 5–10 minutes. Three baseline blood samples were drawn immediately before ingestion of the liquid meal, and further samples were drawn at specific time points from 0–240 minutes. The study also included 2 days of colesevelam + saline and colesevelam + placebo, which will be discussed in future work.

Data collection.

For analyses of GLP-1, glucagon, and Ex9-39, blood was sampled into chilled tubes containing EDTA and a specific DPP-4 inhibitor (3 mmol/l valine-pyrrolidide, 10 μl/ml blood, a gift from Novo Nordisk) and kept on ice until centrifugation. For analysis of insulin, C-peptide, CCK, and total bile acids, blood was collected in dry tubes for coagulation (15 minutes at room temperature). For analysis of acetaminophen, blood was collected in tubes containing heparin. All samples were centrifuged for 20 minute at 1,200 g and 4°C. Plasma samples for GLP-1, glucagon, Ex9-39, CCK, and total bile acids were stored at –20°C, and plasma samples for insulin, C-peptide, and acetaminophen were stored at –80°C. For measurement of plasma glucose, blood was sampled into fluoride tubes and centrifuged immediately at 7,400 g for 2 minutes at room temperature and analyzed bedside. Gallbladder size was evaluated by ultrasound imaging (LOGIQ E9, GE Healthcare) at 6 specific time points (baseline was based on the average of 2 sequential measurements) during the experiment. The gallbladder was measured in 3 dimensions (length and 2 cross-sectional measurements) by a single investigator (MH). Gastric emptying was determined from acetaminophen absorption (65). Gastric emptying is the rate-limiting step for the appearance of orally administered acetaminophen in the blood, and serum acetaminophen time-to-peak and AUC_{60 min} indicate gastric emptying velocity (65). At 6 time points during the experiment, using standardized visual analog scales (VAS), the patients evaluated hunger, satiety, fullness, prospective food consumption, general well-being, nausea, and thirst (66). Before leaving the lab, the patients were served a standardized ad libitum meal and instructed to eat until pleasantly satiated, after which total food intake was noted (66).

Materials.

Metformin (Metformin Actavis tablets containing 500 mg of metformin, Actavis A/S) and acetaminophen (Panodil powder containing 500 mg of acetaminophen, GlaxoSmithKline A/S) were purchased from the Pharmacy of the Capital Region of Denmark. Ex9-39 was purchased from Bachem AG. Ex9-39 was dissolved in sterilized water with 1% human albumin (Plasma Product Unit PSU, Novo Nordisk A/S) and subjected to sterile filtration. Ex9-39 was stored frozen (–20°C) under sterile conditions until the day of the experiment. Ex9-39 was demonstrated to be 99.5% pure by high-performance liquid chromatography. Ex9-39 from the same batch was used for all experimental days.

Laboratory analyzes.

Plasma glucose was measured on a glucose analyzer using the glucose oxidase method (Yellow Springs Instrument model 2300 STAT plus analyzer; YSI Inc.). Plasma insulin and C-peptide were measured using a 2-sided electrochemiluminescence immunoassay (Roche/Hitachi Modular analytics; Roche Diagnostic GmbH) (18). Plasma concentrations of total GLP-1 and CCK were measured by radioimmunoassays as previously described (67, 68). Glucagon was measured with the LINCO assay (MilliporeSigma) because this assay does not cross-react with Ex9-39 (69). Ex9-39 was measured using antibody 3145 raised in rabbits immunized with exendin-4, which shows 100% cross-reactivity with Ex9-39 but only 0.01% cross-reactivity with GLP-1, glucagon, and GIP (69). Serum acetaminophen was measured by the Vitros ACET slide, as previously described (70).

Human tissue collection for ex vivo studies in human ileal and colonic mucosae.

Human ileal and colonic tissue was obtained from patients undergoing bowel resection for cancer or stoma reversal. In the case of resection specimens, samples were obtained from sites at least 10 centimeters away from the tumor location. Specimens from patients that were indicated for any form of inflammatory bowel disease were excluded from this study. Characteristics of the patient cohort are listed in Table 3. The specimens were immediately placed in ice-cold Krebs buffer (NaCl 138 mM, KCl 4.5 mM, CaCl₂ 2.6 mM, NaHCO₃ 4.2 mM, MgCl₂ 1.2 mM, NaH₂PO₄ 1.2 mM, HEPES 10 mM, Glucose 5 mM; MilliporeSigma) and transported to the laboratory for dissection within 15 minutes. The specimens were rinsed with ice-cold Krebs buffer to remove any luminal content and dissected clear of adipose, muscular, and connective tissues. The mucosae were gently dissected off from the submucosae as intact sheets using a stainless-steel spatula, cut into 5-mm pieces, and weighed individually. The mucosal pieces were then transferred to a 96-well plate for secretion assays.

Table 3

Characteristics of the specimen donors

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Secretion experiments.

Mucosal pieces were incubated with 250 μl of buffer (control) or buffer containing test agents in a 96-well plate for 15 minutes as previous (71). While the tissue remains viable for up to 2 hours and displays secretory responses to various ligands within this time frame (unpublished observations), we find that this 15-minute time point provides the greatest degree of fidelity for most reagents tested. The buffer was a modified Krebs buffer described above with the addition of sitagliptin 1 μM, 0.1% fatty acid–free BSA (A1595, MilliporeSigma) at pH 7.4. Following incubation at 37°C in 95% O₂/5% CO₂, supernatants were collected and stored in aliquots at –20°C. Active GLP-1 levels were quantitated using commercially available ELISA kits, according to manufacturer’s instructions (EGLP-35K, MilliporeSigma).

Test agents.

IBMX and FSK (I5879 and F6886, MilliporeSigma) (10 μM each) and 70 mM KCl were used as positive controls. For the 70-mM KCl solution, an equimolar amount of NaCl was removed to maintain osmolarity. The following compounds were purchased from MilliporeSigma: Metformin (PHR1084), Lopinavir (SML1222), Quinine hydrochloride dehydrate (Q1125), and Fluoxetine (F132). The AMPK inhibitor, dorsomorphin, was from MilliporeSigma (171260), as well. Only samples that show positive response to at least 1 positive control (70 mM KCl or 10 μM IBMX/FSK) were included in analysis.

Statistics.

Results are reported as means ± SEM unless otherwise stated. ISR was modeled by application of population-based parameters for C-peptide kinetics to the measured C-peptide concentrations (the deconvolution method) (72). ISR is expressed as picomoles insulin secreted per minute per kilogram body weight. The increased plasma glucose associated with Ex9-39 infusion is, in itself, a driver of insulin secretion. Accordingly, insulin, C-peptide, and ISR data are reported as ratios of the glucose concentration. AUC values were calculated using the trapezoidal rule. If not otherwise stated, AUC values are presented as iAUCs (calculated by subtracting the mean baseline value multiplied by 180 minutes from the total AUC from 0–180 minutes). Gallbladder volume was calculated using the ellipse formula, and gallbladder ejection fraction was calculated as the difference between baseline and the lowest volume divided by the baseline value. Paired, 2-tailed Student’s t tests were used for comparison of the effect of Ex9-39 and saline at baseline, regardless of treatment. One-way repeated measures ANOVA and post hoc Tukey’s multiple comparisons test was used to test for variations and differences of the basal values, AUC, and iAUC between interventions. Two-way ANOVA was performed to illustrate the individual effect of metformin and Ex9-39 on postprandial glucose excursions, as well as a potential interaction between the 2 factors (metformin and Ex9-39). For the ex vivo study, a paired 1-way ANOVA with Fisher’s least significant difference post-hoc test was used for multiple comparisons, and a paired Student’s t tests was used for single comparisons. In case of nonnormally distributed data, logarithmic transformation was performed before statistical testing. All tests were 2-sided, and differences resulting in P < 0.05 were considered significant. Statistical analyzes were carried out using SAS/STAT software version 12.3 (SAS Institute Inc.) or GraphPad Prism software version 6.03 (GraphPad Software Inc.).

Study approval.

The clinical study was performed in accordance with the principles of the Declaration of Helsinki II. All patients provided written informed consent prior to any study activity. For the ex vivo study, patients gave written informed consent for ileum and colon tissue donation from resected small and large intestine at Flinders Medical Centre and Flinders Private Hospital approved by the Southern Adelaide Clinical Human Research Ethics Committee.

Supplemental data:

ICMJE disclosure forms:

Version 1. 12/06/2018

Electronic publication

MH and JSH’s present address are: Novo Nordisk A/S, Bagsværd, Denmark.

Conflict of interest: Funding was received by RLY and DJK from Pfizer Inc. through an Emerging Science Grant. AL and MJ were employees of Pfizer Inc. during this study.

License: Copyright 2018, American Society for Clinical Investigation.

Reference information: JCI Insight. 2018;3(23):e93936. https://doi.org/10.1172/jci.insight.93936.

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