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. 2026 Mar 9;15(3):e250721. doi: 10.1530/EC-25-0721

Metabolic, enterohepatic and gut microbial effects of atorvastatin in healthy men

Martin Thomasen 1, Maria-Anna Misiakou 2,3,*, Simone S Li 2,4, Mari Cristina Rodriguez de Evgrafov 2,†, Mads B Lynggaard 1, Martin L Kårhus 1, Andreas Brønden 5,6, Jonatan Kornholt 5,*, Oscar Chávez-Talavera 7, Anne Tailleux 7, Bart Staels 7, Amandine Descat 7, Bolette Hartmann 8, Nicolai J Wewer Albrechtsen 9, Jens F Rehfeld 10, Jens J Holst 8,11, Tina Vilsbøll 6,12, Anne-Marie Ellegaard 1,8, Morten O A Sommer 2,✉, David P Sonne 5,6, Filip K Knop 1,6,*,✉
PMCID: PMC12978631  PMID: 41738774

Abstract

Objective

Statins are low-density lipoprotein cholesterol-lowering drugs that are highly effective in the prevention of cardiovascular disease and death. Evidence that statin therapy increases the risk of type 2 diabetes is accumulating, but the mechanism behind this phenomenon remains obscure.

Design

A clinical, randomised, placebo-controlled, double-blind, crossover study.

Methods

Here, we investigated the effect of atorvastatin on fasting and postprandial circulating concentrations of glucose, gluco-regulatory hormones, bile acid profiles and gut microbiota composition. Fifteen healthy men came in for a mixed meal test following 14 days of treatment with atorvastatin (40 mg once-daily during week one and 80 mg once-daily during week two) or placebo.

Results

Treatment with atorvastatin did not affect postprandial plasma glucose or insulin concentrations, but basal and postprandial concentrations of glucagon were increased compared with placebo. Postprandial plasma concentrations of the gut-derived incretin hormones glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 were increased after atorvastatin treatment compared with placebo. In addition, postprandial concentrations of taurine-conjugated primary bile acids increased, whereas glycine-conjugated secondary bile acids decreased. Microbiota composition was not affected by atorvastatin treatment.

Conclusions

Atorvastatin treatment did not alter glucose or insulin concentrations, nor did it alter gut microbiota composition. However, we found that atorvastatin treatment increased fasting and postprandial glucagon concentrations, which may point to hyperglucagonaemia as a possible link between statin treatment and type 2 diabetes.

Clinicaltrials.gov

NCT03018444.

Significance statement

This randomised, placebo-controlled, double-blind, crossover study reveals that short-term high-dose atorvastatin treatment increases fasting and postprandial glucagon levels and alters amino acid and bile acid profiles without affecting glucose, insulin or gut microbiota in healthy men. These findings suggest hyperglucagonaemia as a potential mechanistic contributor to the increased risk of type 2 diabetes associated with statin therapy.

Keywords: type 2 diabetes, atorvastatin, glucagon, glucagon-like peptide 1, amino acids, bile acids, gut microbiome

Introduction

By decreasing circulating levels of low-density lipoprotein (LDL) cholesterol, a key player in the formation of atherosclerotic plaques, statin therapy saves millions of people from cardiovascular morbidity and mortality (1). Statins lower circulating LDL cholesterol levels by inhibiting de novo cholesterol formation by competitively inhibiting the enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which results in an upregulation of LDL cholesterol receptors in hepatocytes and a consequential increased uptake of LDL cholesterol from the circulation (2, 3, 4). The effectiveness of statins in reducing the risk of cardiovascular morbidity and mortality is proportional to the absolute LDL cholesterol reduction (5, 6, 7). Statins are considered effective with overall benign adverse effects, and their use has increased in the past decades (8, 9). However, accumulating evidence shows that statin use increases the risk of incident diabetes (10, 11) and progression of diabetes (12). A meta-analysis including 13 randomised controlled trials (91,140 participants) documented a 9% increased risk of incident diabetes compared with placebo (10). Several observational studies reported similar associations (13, 14, 15, 16, 17). Furthermore, a retrospective cohort study revealed that patients with type 2 diabetes treated with statins are at an increased risk of diabetes progression (12). Moreover, a recent meta-analysis of randomised controlled statin trials showed that the risk of incident diabetes is dose-dependent (18). The mechanisms underlying this off-target diabetogenic effect of statins remain obscure. In vitro studies and animal models have indicated that statins may induce insulin resistance because of decreased glucose transporter type 4 (GLUT4) expression and/or perturbations of the insulin signalling cascade (19, 20, 21). Others have proposed that statins induce beta-cell dysfunction by blockage of L-type calcium channels (22, 23, 24) or by enhancing the beta-cell potassium current (23). Bile acids, synthesised from cholesterol, constitute another potential actor in the off-target metabolic effects of statins. Bile acids have gained renewed attention as endogenous hormones and metabolic regulators, following the discovery that they are ligands for the Takeda G protein-coupled receptor 5 (TGR5) (25, 26) and the nuclear farnesoid X receptor (FXR) (27, 28). Stimulating these receptors, expressed in the enteroendocrine L-cells, affects the secretion of the glucose-lowering and satiety-promoting gut incretin hormone glucagon-like peptide 1 (GLP-1) (29, 30, 31). Furthermore, individuals with insulin resistance and patients with type 2 diabetes are characterised by altered fasting and postprandial bile acid composition (32, 33, 34). Finally, bile acids have strong and bidirectional interactions with the gut microbiota (35, 36, 37) and several association studies have linked gut microbial dysbiosis to insulin resistance and type 2 diabetes (38, 39, 40, 41).

Human studies specifically investigating the metabolic off-target effects of statins are lacking. Here, we investigated the effects of a 14-day high-dose atorvastatin treatment on glucose, gluco-metabolic hormones, bile acids and gut microbiota in 15 healthy young men in a randomised, placebo-controlled, double-blind, crossover study. We hypothesised that atorvastatin treatment would result in a reduced postprandial GLP-1 plasma level due to altered bile acid profiles compared to placebo.

Methods

Participants

Fifteen healthy men were recruited (Supplementary Fig. S1 (see section on Supplementary materials given at the end of the article), Table 1). The inclusion criteria were body mass index (BMI) 18.5–35 (kg/m2), glycated haemoglobin (HbA1c) < 43 mmol/mol, fasting plasma glucose < 6 mmol/L, normal haemoglobin and Caucasian ethnicity. Exclusion criteria encompassed diagnosed diabetes, first-degree relatives with diabetes, liver disease, gastrointestinal disease, previous intestinal resection, cholecystectomy or any major abdominal surgery, reduced kidney function, taking any kind of medicine on a regular basis, intake of antibiotics two months prior to the study and active or recent malignant disease.

Table 1.

Baseline characteristics of study participants.

Parameter Value
Men/women (n/n) 15/0
Age (years) 26 (21–35)
BMI (kg/m2) 25 (22–31)
HbA1c (%)/(mmol/mol) 4.8 (4.3–5.0)/29 (24–31)
Fasting plasma glucose (mmol/L) 5.0 (4.3–5.5)
ALT (U/L) 32 (22–44)
Total cholesterol (mmol/L) 4.3 (3.3–5.5)
LDL cholesterol (mmol/L) 2.4 (1.3–3.2)
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Mean values and ranges in brackets are shown. BMI, body mass index; HbA1c, heamoglobin A1c; ALT, alanine aminotransferase; LDL, low-density lipoprotein.

Study design

This was a randomised, placebo-controlled, double-blind, crossover trial. Participants were randomised to receive either atorvastatin treatment or placebo in the first 14-day treatment period and then, after a washout period of at least 14 days, crossed over to the opposite treatment for the second 14-day treatment period. For randomisation of treatment sequence (placebo followed by atorvastatin vs atorvastatin followed placebo), the website https://randomizer.org/ was used by an independent researcher otherwise not involved in the project. Active treatment consisted of atorvastatin 40 mg once daily from day 1 to 7 and 80 mg once daily from day 8 to 14. The participants were instructed to take the agent before bedtime and came in for an experimental day the morning after the last day of each treatment period; with stool sample collection 1–2 days prior. Participants were instructed to maintain a similar diet during both treatment periods and asked to maintain their regular daily living activities throughout the study. Furthermore, they were instructed to abstain from excessive intake of alcohol and fat and high-intensity exercise three days before each experimental day. Participants arrived at the laboratory in the morning after an overnight (10 h) fast (including water, coffee, medicine and use of tobacco). We adopted a range of exploratory endpoints, including postprandial plasma/serum responses of gluco-metabolic hormones, including GLP-1, glucose-dependent insulinotropic polypeptide (GIP), glucagon, cholecystokinin (CCK) and gastrin; glucose tolerance; postprandial bile acid composition; lipid composition; fasting amino acid composition; gallbladder and gastric emptying; and gut microbiota composition. The sample size was calculated based on plasma GLP-1 responses from a previous study (42) and based on a power of 80% and an estimated minimal relevant difference of 30%.

Experimental procedures

Each participant received equipment and instructions (oral and written) on stool sample collection prior to the experimental days. Stool samples were collected by the participant at home and immediately stored in a home freezer. On experimental days, participants arrived at our clinical research facility in the morning after an overnight fast, bringing the frozen stool sample collected 1–2 days prior in an insulated cooling bag containing the sample and two frozen freezer blocks for immediate storage in a −80°C freezer. Participants were semi-recumbently positioned in a hospital bed, and a cannula was placed in a cubital vein for collection of arterialised blood samples (cannulated forearm was wrapped in a heating pad (∼45°C) throughout the experiment). After baseline sampling at time point 0 min, participants received a liquid mixed meal (200 mL, 1,260 kJ (36.8 g carbohydrate, 11.6 g protein and 12.0 g of lipid), Nutricia, Danone, Denmark) mixed with 1,500 mg paracetamol (for evaluation of gastric emptying) dissolved in 100 mL water, which was ingested over 5 min. Blood samples were collected at time points −30, −15, 0, 10, 20, 30, 45, 60, 75, 90, 120, 150, 180, 210 and 240 min. For bedside analysis of plasma glucose, blood was collected in sodium fluoride-coated tubes and centrifuged immediately for 30 s at 7,500 g at room temperature. For the analyses of GLP-1, GIP, glucagon, amino acids, gastrin, CCK, fibroblast growth factor 19 (FGF19) and bile acids in plasma, blood was collected into chilled ethylenediaminetetraacetic acid (EDTA) tubes coated with a specific dipeptidyl peptidase 4 (DPP-4) inhibitor (valine-pyrrolidide, final concentration of 0.01 mmol/L, a gift from Novo Nordisk A/S, Denmark). EDTA tubes were kept on ice until centrifuged. For the analyses of insulin, C-peptide and 7α-hydroxy-4-cholesten-3-one (C4) in serum, blood was collected in dry tubes with serum separator gel and clot activator and left for 20 min at room temperature for coagulation. For analyses of paracetamol and lipid profile in plasma, blood was collected into lithium-heparin tubes. All tubes were centrifuged for 15 min at 2,900 g and 4°C. Plasma and serum samples were stored at either −20°C or −80°C until analysis.

Analyses of blood samples

Analyses of blood samples are described in detail in the supplementary material.

Calculations and statistical analysis of anthropomorphic and biochemical data

Descriptive statistics were used to characterise the participants’ age and BMI as well as glycated HbA1c, cholesterol levels and alanine aminotransferase (ALAT) at the time of screening. Area under the curve (AUC) and baseline-subtracted AUC (bsAUC) values were calculated using the trapezoidal rule. All continuous data were analysed with ordinary least-squares linear regression with subject, treatment and period as factors (43). Least-squares means (classical Yates’ contrasts) were computed to assess between-group differences (atorvastatin vs placebo). Based on the recommendations by Senn (43), carry-over effects were not tested due to sufficient washout period. The assumptions of a Gaussian distribution of residuals and homogeneity of variances were assessed visually by drawing histograms, residual plots and probability plots. If assumptions could not be met, continuous variables were transformed using appropriate transformations. Non-continuous data, i.e. time to peak values, were analysed with non-parametric methods using the Hodges–Lehmann estimator with accompanying 95% confidence intervals (43). A two-sided P value ≤0.05 was used to indicate significant differences. No correction for multiple testing was performed as this study was mainly exploratory in nature. Statistical analyses were carried out using RStudio, version 1.4.1103, with R, version 3.6.1, with tidyverse (44) for utility functions. Plots were drawn using GraphPad Prism, version 8.30, for Windows.

Analysis of gut microbiome in faecal samples

Extraction of faecal DNA, metagenomic sequencing, and processing and taxonomic and functional profiling of stool metagenomes are described in detail in the supplementary material.

Results

Baseline participant characteristics

We screened 17 young men for inclusion in the study; 15 were eligible for participation and all completed the study (Supplementary Fig. S1). The mean age was 26 years (range 21–35), and their mean BMI and mean fasting plasma glucose were 25 kg/m2 (range 22–31) and 5.0 mmol/L (range 4.3–5.5), respectively. The participant characteristics are shown in Table 1.

Fasting lipid profiles were altered by atorvastatin treatment

Compared with placebo, atorvastatin treatment reduced plasma levels of total cholesterol −1.5 mmol/L (95% CI: −1.8; −1.2, P < 0.001), LDL cholesterol −1.3 mmol/L (95% CI: −1.5; −1.0, P < 0.001), very-low-density lipoprotein (VLDL) cholesterol −0.1 mmol/L (−0.2; 0.0, P = 0.007) and triglycerides −0.3 mmol/L (95% CI: −0.5; −0.1, P = 0.009) but did not alter plasma high-density lipoprotein (HDL) cholesterol levels (Table 2).

Table 2.

Overview of plasma, serum and ultrasonographic measurements.

Placebo Atorvastatin Mean of differences (atorvastatin – placebo) P value
Fasting lipid profile
 Total cholesterol (mmol/L) 3.9 (3.6; 4.3) 2.3 (2.1; 2.5) −1.5 (−1.8; −1.2) <0.001
 LDL cholesterol (mmol/L) 2.4 (2.0; 2.7) 1.0 (0.8; 1.3) −1.3 (−1.5; −1.0) <0.001
 HDL cholesterol (mmol/L) 1.2 (1.0; 1.3) 1.1 (0.9; 1.2) −0.1 (−0.2; 0.1) 0.271
 VLDL cholesterol (mmol/L) 0.4 (0.3; 0.4) 0.2 (0.2; 0.3) −0.1 (−0.2; 0.0) 0.007
 Triglycerides (mmol/L) 0.8 (0.6; 0.9) 0.5 (0.4; 0.6) −0.3 (−0.5; −0.1) 0.009
Glucose
 Fasting (mmol/L) 4.9 (4.8; 5.0) 4.9 (4.6; 5.1) 0.0 (−0.1; 0.2) 0.905
 Peak (mmol/L) 6.6 (6.3; 6.9) 6.6 (6.3; 7.1) 0.2 (−0.1; 0.6) 0.179
 Time to peak (min) 30 (30; 37.5) 30 (30; 45) 5 (−7.5; 12.5) 0.400
 bsAUC0–240 (mmol/L × min) 50 (5; 78) 69 (37; 110) 25 (−12; 63) 0.167
 AUC0–240 (mmol/L × min) 1,228 (1,140; 1,253) 1,226 (1,182; 1,298) 28 (−9; 64) 0.126
Insulin
 Fasting (pmol/L) 50.4 (34.3; 60.5) 41.1 (32.1; 62.7) 4.7 (−6.0; 15.3) 0.362
 Peak (pmol/L) 484.6 (411.4; 701.0) 433.4 (358.4; 588.1) 7.2 (−126.6; 141.1) 0.909
 Time to peak (min) 30 (30; 37.5) 30 (30; 45) 5 (0; 20) 0.230
 bsAUC0–240 (pmol/L × min) 18,078 (15,060; 23,198) 17,208 (15,712; 29,115) 3,282 (−1,989; 8,553) 0.202
 AUC0–240 (pmol/L × min) 30,155 (25,088; 36,008) 28,401 (24,471; 40,743) 4,402 (−2,082; 10,886) 0.166
C-peptide
 Fasting (pmol/L) 366 (274; 509) 350 (301; 489) 19 (−36; 74) 0.468
 Peak (pmol/L) 1,754 (1,489; 1,946) 1,566 (1,345; 2,075) 31 (−131; 192) 0.687
 Time to peak (min) 45 (30; 45) 45 (30; 60) 0 (−7.5; 7.5) 0.540
 bsAUC0–240 (pmol/L × min) 84,290 (71,365; 101,470) 98,141 (69,159; 114,079) 7,883 (−8,090; 23,857) 0.306
 AUC0–240 (pmol/L × min) 173,266 (14,386; 205,898) 182,431 (14,150; 220,500) 12,433 (−5,968; 30,835) 0.168
Glucagon
 Fasting (pmol/L) 5.3 (3.3; 7.5) 8.3 (6.3; 9.3) 2.2 (0.7; 3.7) 0.008
 Peak (pmol/L) 9.0 (8.0; 13.0) 13.0 (9.5; 14.5) 1.9 (0.6; 3.2) 0.006
 Time to peak (min) 45 (30; 60) 45 (20; 75) 2.5 (−70; 30) 0.870
 bsAUC0–240 (pmol/L × min) 360 (256; 502) −197 (−280; 466) −246 (−585; 93) 0.141
 AUC0–240 (pmol/L × min) 1,647 (1,331; 2,171) 1,737 (1,560; 2,513) 273 (118; 429) 0.002
GIP
 Fasting (pmol/L) 7.0 (4.2; 9.7) 12.0 (6.3; 15.7) 4.4 (2.0; 6.8) 0.002
 Peak (pmol/L) 69.0 (58.5; 84.0) 76.0 (67.0; 93.5) 7.0 (−2.8; 16.8) 0.147
 Time to peak (min) 30 (30; 45) 45 (30; 52.5) 7.5 (−7.5; 22.5) 0.280
 bsAUC0–240 (pmol/L × min) 5,648 (4,345; 6,889) 5,127 (4,776; 7,090) −185 (−1,189; 820) 0.698
 AUC0–240 (pmol/L × min) 7,717 (6,601; 9,020) 7,878 (7,478; 10,170) 876 (245; 1,507) 0.010
GLP-1
 Fasting (pmol/L) 7.3 (5.3; 10.8) 9.3 (8.2; 12.0) 1.6 (−0.9; 4.1) 0.182
 Peak (pmol/L) 24.0 (20.0; 28.5) 25.0 (20.5; 32.5) 1.9 (−0.8; 4.7) 0.149
 Time to peak (min) 60 (45; 105) 60 (37.5; 135) 7.5 (−20; 30) 0.540
 bsAUC0–240 (pmol/L × min) 1,211 (756; 1,911) 944 (811; 1,256) −33 (−649; 584) 0.911
 AUC0–240 (pmol/L × min) 3,031 (2,602; 3,662) 3,494 (3,099; 3,743) 360 (3; 718) 0.049
Gastrin
 Fasting (pmol/L) 7.0 (5.0; 8.0) 7.3 (5.0; 8.8) 0.5 (−0.3; 1.0) 0.262
 Peak (pmol/L) 13.0 (8.0; 14.5) 10.0 (8.0; 14.0) −0.5 (−1.6; 0.6) 0.375
 Time to peak (min) 10 (10; 37.5) 20 (10; 45) 7.5 (−10; 15) 0.230
 bsAUC0–240 (pmol/L × min) 358 (229; 534) 363 (180; 510) −91 (−338; 156) 0.440
 AUC0–240 (pmol/L × min) 2,016 (1,463; 2,516) 2,068 (1,526; 2,537) −3 (−147; 142) 0.970
CCK
 Fasting (pmol/L) 1.0 (0.7; 1.6) 1.4 (1.0; 1.6) 0.3 (−0.1; 0.6) 0.150
 Peak (pmol/L) 6.3 (4.6; 7.9) 7.6 (4.7; 8.7) 1.1 (−1.0; 3.2) 0.291
 Time to peak (min) 20 (10; 20) 10 (10; 20) −5 (−10; 5) 0.400
 bsAUC0–240 (pmol/L × min) 254 (202; 444) 215 (172; 356) −39 (−140; 61) 0.416
 AUC0–240 (pmol/L × min) 513 (457; 673) 564 (481; 714) 24 (−47; 95) 0.484
Total amino acids
 Fasting (μmol/L) 2,071 (1,684; 2,111) 2,031 (1,893; 2,131) 137 (−65; 338) 0.166
Paracetamol
 Peak (mmol/L) 0.094 (0.082; 0.107) 0.093 (0.083; 0.105) 0.000 (−0.007; 0.008) 0.925
 Time to peak (min) 75 (75; 90) 90 (75; 120) 0 (−15; 22.5) 0.610
FGF19
 Fasting (pg/mL) 61.8 (46.5; 97.1) 88.0 (61.8; 138.1) 31.7 (−38.7; 102.1) 0.349
 Peak (pg/mL) 288.6 (176.1; 356.1) 220.3 (154.1; 363.9) −9.4 (−114.0; 95.1) 0.848
 Time to peak (min) 210 (180; 240) 210 (165; 210) −15 (−75; 15) 0.230
 bsAUC0–240 (pg/mL × min) 16,744 (5,538; 22,714) 12,865 (5,185; 19,748) −2,552 (−12,834; 7,729) 0.601
 AUC0–240 (pg/mL × min) 33,205 (21,142; 50,071) 34,167 (24,748; 47,425) 5,054 (−13,094; 23,202) 0.558
C4
 Fasting (nmol/L) 33.1 (16.2; 42.3) 17.1 (12.0; 29.3) −14.3 (−26.8; −1.8) 0.028
 Peak (nmol/L) 39.5 (31.8; 55.8) 18.4 (16.2; 39.4) −22.3 (−40.7; −3.8) 0.022
 Time to peak (min) 240 (15; 240) 240 (15; 240) 0 (−120; 15) 0.690
 bsAUC0–240 (nmol/L × min) −333 (−799; 1,645) −17 (−935; 514) −883 (−2,460; 693) 0.248
 AUC0–240 (nmol/L × min) 6,756 (4,679; 10,339) 3,309 (3,098; 5,602) −4,317 (−7,593; −1,040) 0.013
Gallbladder
 Fasting (cm3) 28.6 (19.4; 44.2) 27.1 (18.0; 40.1) −2.8 (−7.6; 2.1) 0.238
 Maximal EF (%) 67.7 (59.8; 73.6) 65.8 (59.1; 78.2) 0.9 (−6.2; 8.0) 0.783
 Time to maximal EF (min) 55 (55; 55) 55 (55; 55) 0 (−17.5; 2.5) 0.400
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Plasma, serum and ultrasonographic measurements during standardised mixed meal test (meal served at time 0 min) in 15 healthy men following double-blind 14-day placebo treatment and 14-day atorvastatin treatment in randomised order and separated by a 14-day washout period. Median and interquartile ranges in brackets are shown in placebo and atorvastatin columns. Mean of differences between atorvastatin and placebo are shown with 95% confidence intervals in brackets. P values shown are from comparison of atorvastatin and placebo treatments. AUC, area under the curve; bsAUC, baseline subtracted area under the curve; LDL, low-density lipoprotein; HDL, high-density lipoprotein; VLDL, very-low-density lipoprotein; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide 1; CCK, cholecystokinin; FGF19, fibroblast growth factor 19; C4, 7α-hydroxy-4-cholesten-3-one, EF; ejection fraction.

Atorvastatin had no impact on circulating glucose, insulin and C-peptide levels but increased glucagon levels

Plasma glucose concentrations in the fasted state and postprandial plasma glucose excursions were similar after atorvastatin treatment and placebo (Fig. 1, Table 2). Basal concentrations of insulin and C-peptide, respectively, were comparable following atorvastatin and placebo (Fig. 1, Table 2). Likewise, postprandial insulin and C-peptide responses were unaffected by atorvastatin treatment (Fig. 1, Table 2). Compared with placebo, atorvastatin treatment increased plasma glucagon concentrations in the fasted state by 2.2 pmol/L (95% CI: 0.7; 3.7, P = 0.008) and the postprandial plasma glucagon AUC by 273 pmol/L × min (95% CI: 118; 429, P = 0.002) (Fig. 1, Table 2). The postprandial increase in glucagon following atorvastatin treatment was driven by the increased fasting levels as the bsAUC was not different compared with placebo (Table 2). Atorvastatin treatment did not affect gastric emptying of the liquid meal as assessed by plasma paracetamol excursions (Supplementary Fig. S2, Table 2).

Figure 1.

Figure 1

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Glucose, insulin, C-peptide and glucagon. Plasma/serum concentrations of glucose (A), insulin (C), C-peptide (E) and glucagon (G) during a standardised mixed meal test (meal served at time 0 min) in 15 healthy men following double-blind 14-day placebo treatment (white circles, dashed line) and 14-day atorvastatin treatment (black squares, unbroken line) in randomised order and separated by a 14-day washout period and corresponding area under the curve (AUC) (0–240 min) (B, D, F, H). Mean values ± standard error of the mean are shown on line graphs (A, C, E, G), and median ± interquartile range is shown on column bar graphs (B, D, F, H). Least-squares means were calculated, and significant differences (P ≤ 0.01) are marked with asterisks (**).

Fasting amino acid profiles were altered by atorvastatin treatment

The plasma concentration of total amino acids in the fasted state was unaltered by atorvastatin treatment compared with placebo (Table 2). Analysis of individual plasma amino acid concentrations showed that atorvastatin treatment increased basal concentrations of glutamic acid, glycine, methionine, serine and threonine (Fig. 2, Supplementary Table S1).

Figure 2.

Figure 2

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Amino acids. Plasma concentrations of amino acids in the fasted state in 15 healthy men following double-blind 14-day placebo treatment (white bars) and atorvastatin treatment (black bars) in randomised order and separated by a 14-day washout period. Median values are shown. Least-squares means were calculated, and significant differences (P ≤ 0.05) and (P ≤ 0.01) are marked with asterisks (*) and (**), respectively, and these amino acids are shown in bold.

Atorvastatin treatment increased circulating GIP and GLP-1 levels, whereas CCK and gastrin levels were unaffected

Compared with placebo, atorvastatin treatment increased plasma GIP concentrations in the fasted state by 4.4 pmol/L (95% CI: 2.0; 6.8, P = 0.002) (Table 2) and the postprandial AUC for plasma GIP by 876 pmol/L × min (95% CI: 245; 1,507, P = 0.010) (Fig. 3, Table 2). The increased GIP AUC appeared driven by the increased fasting concentration as the bsAUC during atorvastatin treatment was not different from that observed during placebo treatment (Table 2). Compared with placebo, basal plasma GLP-1 concentrations were unaffected by atorvastatin treatment, whereas the postprandial AUC for plasma GLP-1 was increased by 360 pmol/L × min (95% CI: 3; 718, P = 0.049) (Fig. 3, Table 2). Basal and postprandial plasma gastrin and CCK concentrations were unaffected by atorvastatin treatment (Fig. 3, Table 2).

Figure 3.

Figure 3

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GIP, GLP-1, gastrin and CCK. Plasma concentrations of GIP (A), GLP-1 (C), gastrin (E) and CCK (G) during a standardised mixed meal test (meal served at time 0 min) in 15 healthy men following double-blind 14-day placebo treatment (white circles, dashed line) and 14-day atorvastatin treatment (black squares, unbroken line) in randomised order and separated by a 14-day washout period and corresponding area under the curve (AUC) (0–240 min) (B, D, F, H). Mean values ± standard error of the mean are shown on line graphs (A, C, E, G), and median ± interquartile range is shown on column bar graphs (B, D, F, H). Least-squares means were calculated, and significant differences (P ≤ 0.05) and (P ≤ 0.01) are marked with asterisks (*) and (**), respectively. GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide 1; and CCK, cholecystokinin.

Atorvastatin treatment reduced bile acid synthesis but had no effect on gallbladder volume or dynamics

Compared with placebo, atorvastatin treatment reduced fasting and peak plasma concentrations and the AUC of the bile acid synthesis marker C4 by −14.3 nmol/L (95% CI: −26.8; −1.8, P = 0.028), −22.3 nmol/L (95% CI: −40.7; −3.8, P = 0.022) and −4,317 nmol/L × min (95% CI: −7,593; −1,040, P = 0.013), respectively (Fig. 4, Table 2). Compared with placebo, gallbladder volume during fasting and postprandial gallbladder dynamics (maximal ejection fraction and time to maximal ejection fraction) were unaffected by atorvastatin treatment (Supplementary Fig. S2, Table 2). Fasting and postprandial concentrations of FGF19 were unaltered by atorvastatin treatment (Fig. 4, Table 2).

Figure 4.

Figure 4

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Total bile acids, FGF19 and C4. Plasma/serum concentrations of total bile acids (A), FGF19 (C) and C4 (E) during a standardised mixed meal test (meal served at time 0 min) in 15 healthy men following double-blind 14-day placebo treatment (white circles, dashed line) and 14-day atorvastatin treatment (black squares, unbroken line) in randomised order and separated by a 14-day washout period and corresponding area under the curve (AUC) (0–240 min) (B, D, F). Mean values ± standard error of the mean are shown on line graphs (A, C, E), and median ± interquartile range is shown on column bar graphs (B, D, F). Least-squares means were calculated, and significant differences (P ≤ 0.05) are marked with asterisk (*). FGF19, fibroblast growth factor 19; C4, 7α-hydroxy-4-cholesten-3-one.

Atorvastatin treatment exerted distinct effects on individual bile acids in the circulation after meal ingestion

The fasting levels of total and individual bile acids were unchanged by atorvastatin treatment compared with placebo (Supplementary Table S2). The total bile acid AUC was unaltered by atorvastatin treatment compared with placebo (Figs 4 and 5, Supplementary Table S3). Separating total bile acids into their conjugates revealed that atorvastatin treatment, compared with placebo, caused a small increase in postprandial concentrations of taurine conjugates, whereas postprandial glycine conjugates tended to be lowered by atorvastatin treatment (Fig. 5, Supplementary Table S3). Accordingly, atorvastatin treatment reduced the ratio between postprandial glycine and taurine conjugates (Supplementary Table S4). Atorvastatin treatment did not alter the ratio of postprandial conjugated to unconjugated bile acids compared with placebo (Supplementary Table S4). Compared with placebo, atorvastatin treatment did not alter total primary bile acids or the individual primary bile acids (total cholic acid (CA), chenodeoxycholic acid (CDCA) and hyocholic acid (HCA)) in the postprandial state but increased the concentrations of primary bile acid taurine conjugates (taurine-conjugated CA, CDCA and HCA) (Fig. 5, Supplementary Table S3). Postprandial total secondary bile acids decreased after atorvastatin treatment, which was driven by reductions in glycine-conjugated deoxycholic acid (DCA), ursodeoxycholic acid (UDCA) and lithocholic acid (LCA), respectively (Fig. 5, Supplementary Table S3). Despite the atorvastatin-induced decrease in postprandial secondary bile acids, the ratio of primary to secondary bile acids was unaltered by atorvastatin treatment (Supplementary Table S4).

Figure 5.

Figure 5

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Individual bile acids. Postprandial area under the curve (AUC) (0–240 min) of total bile acids (TBAs), cholic acid (CA), chenodeoxycholic acid (CDCA), hyocholic acid (HCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA) and lithocholic acid (LCA) and their corresponding glycine (g) and taurine (t) conjugates as well as total primary bile acids (1°) and total secondary bile acids (2°) during a standardised mixed meal test (meal served at time 0 min) in 15 healthy men following double-blind 14-day placebo treatment (white bars) and 14-day atorvastatin treatment (white bars with diagonal fill) in randomised order and separated by a 14-day washout period. Median ± interquartile ranges are shown. Least-squares means were calculated, and significant differences (P ≤ 0.05) and (P ≤ 0.01) are marked with asterisks (*) and (**), respectively.

Atorvastatin treatment did not modulate the human gut microbiome

Shotgun sequencing of metagenomic DNA extracted from stool samples produced approximately 3.47 ± 0.25 Gbp per sample (Supplementary Table S5). Taxonomic profiling revealed 267 microbial species across our study cohort, 90 of which were putative (Supplementary Table S6). Of the known phyla, no differences were found between paired atorvastatin and placebo samples (Wilcoxon signed rank test P > 0.05) (Supplementary Fig. S3, Supplementary Table S7). Substantial inter-individual variation of the most prevalent species (including Prevotella copri) was found across participants (Fig. 6), and community diversity and composition were unchanged in response to atorvastatin treatment (PERMANOVA P > 0.05) (Supplementary Figs S4 and S5). When extending the analysis to include functional metagenomic profiling using the Integrated Gene Catalogue (ICG), atorvastatin induced no changes to the functional potential of the gut microbiome (PERMANOVA P > 0.05). Furthermore, differential analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthology (KO) abundances revealed no shifts between atorvastatin and placebo groups (Wilcoxon signed rank test, FDR < 0.05), including genes involved in bile acid metabolism (Supplementary Fig. S6, Supplementary Table S8).

Figure 6.

Figure 6

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Microbiome taxonomic compositions of stool samples collected in the study. The mean relative abundance of the top 18 taxa present in the gut microbiome of 15 participants following placebo and atorvastatin treatment, respectively, is visualised; the remainder are present in grey. Bifidobacterium catenulatum complex is an abbreviation of Bifidobacterium catenulatum–Bifidobacterium pseudocatenulatum complex.

Discussion

This randomised, placebo-controlled, double-blind, crossover study was designed to shed light on the mechanisms underlying the association between statin therapy and development of diabetes (10, 11). We show that 14-day high-dose atorvastatin therapy in young, healthy men resulted in hyperglucagonaemia, known to be a central player in the development of type 2 diabetes (45). We also show that atorvastatin treatment increases circulating levels of the glucose-lowering gut incretin hormones GIP and GLP-1, which may counteract detrimental glycaemic effects of atorvastatin-induced hyperglucagonaemia. Finally, we show that atorvastatin treatment results in a robust reduction in bile acid synthesis, as assessed by circulating C4 coinciding with reduced postprandial plasma concentrations of mostly secondary bile acid glycine conjugates, with no or negligible effects on gut microbiota composition. Limitations of the study include its relatively small sample size, exclusive healthy young Caucasian male study population and relatively short duration of treatment; thus, the findings of the study should be viewed as hypothesis-generating.

In a recent cross-sectional study, individuals with prediabetes taking atorvastatin (n = 26) exhibited attenuated suppression of plasma glucagon levels during an oral glucose tolerance test and, thus, hyperglucagonaemia vs individuals with prediabetes not taking statin therapy (n = 24) (46). The study’s cross-sectional nature prevented a causal relationship between changes in plasma glucagon and atorvastatin therapy from being established. Nevertheless, the authors also reported that atorvastatin exposure of mouse glucagonoma α-TC1 (clone 6) cells impaired insulin-induced suppression of glucagon release and proglucagon gene transcription, thus supporting a direct link between atorvastatin therapy and glucagon secretion (46). Interestingly, glucagon receptor antagonism has been shown to increase circulating levels of LDL cholesterol (47, 48), which has been linked to increased cholesterol absorption (49) and upregulation of proprotein convertase subtilisin/kexin type 9 (50). In conjunction with our findings, this suggests an interplay between LDL cholesterol and glucagon that warrants further investigation. Amino acids and glucagon are linked in a mutual feedback cycle, referred to as the liver–alpha cell axis (51). Most amino acids are glucagonotropic in vivo and glucagon increases ureagenesis, and thus, amino acid turnover (51, 52). Here, we find that glutamic acid, glycine, methionine, serine and threonine levels were elevated following atorvastatin treatment. The mechanism by which these amino acids become elevated during atorvastatin treatment cannot be elucidated from the present data, but the increased circulating concentrations may play a role in the observed fasting hyperglucagonaemia. Whether atorvastatin-induced hyperglucagonaemia as seen here in healthy young men at a low risk of diabetes would induce hyperglycaemia in people at a high risk of diabetes is currently uncertain.

GIP and GLP-1, the two incretin hormones, potentiate glucose-stimulated insulin secretion and contribute to postprandial glucose tolerance in healthy individuals (53). Furthermore, GIP and GLP-1 have opposing effects on pancreatic glucagon secretion, with GIP stimulating glucagon secretion at normal and low blood glucose levels and GLP-1 potentiating glucose-induced inhibition of glucagon secretion (54, 55). GLP-1 also increases satiety and reduces food intake, and several GLP-1 analogues are approved for the treatment of type 2 diabetes and obesity (56). To our knowledge, the effect of atorvastatin on circulating levels of incretin hormones in humans has not been investigated previously. The atorvastatin-induced elevations of circulating incretin hormones observed here are relatively small, and the study design does not allow inference on incretin-derived effects. However, we speculate that they may counteract hyperglucagonaemia-induced elevation of blood glucose levels.

Bile acids have been hypothesised to play a role in the pathophysiology of type 2 diabetes (57). Enteroendocrine L-cells express bile acid-activated receptors (29, 30, 31), and enteral administration of the primary bile acid CDCA has been shown to increase circulating levels of GLP-1 and glucagon in individuals with type 2 diabetes and in individuals with normal glucose tolerance (58, 59). Furthermore, individuals with type 2 diabetes have an altered postprandial bile acid profile (33) and, following the Roux-en-Y gastric bypass procedure, individuals have increased fasting levels of total bile acids (60). Since cholesterol serves as a backbone for bile acid synthesis, we hypothesised that an atorvastatin-induced reduction in de novo cholesterol formation via inhibition of HMG-CoA reductase would lead to perturbations of the enterohepatic circulation of bile acids, including postprandial plasma levels. We show, for the first time, that atorvastatin treatment almost halves bile acid synthesis as assessed by circulating C4 and results in reduced postprandial plasma concentrations of secondary bile acid glycine conjugates, increases postprandial plasma concentrations of primary bile acid taurine conjugates and lowers the postprandial ratio of glycine conjugates to taurine conjugates. The atorvastatin-induced postprandial bile acid profile changes, however, do not resemble the changes observed in individuals with type 2 diabetes, who exhibit elevated postprandial levels of glycine conjugated and unconjugated secondary bile acids and elevated postprandial levels of glycine-conjugated CA (33). Bile acid synthesis and bile acid profiles generally exhibit relatively high inter-individual variations (61, 62). With our current understanding of bile acids in relation to gluco-metabolic disease, it is difficult to ascertain whether the observed changes are unfavourable, beneficial or of no consequence to metabolic status.

The role of the human gut microbiome in various diseases and its response to their pharmacological treatments is still unclear, with most studies highlighting associations but rarely deciphering causality (63). Some non-antibiotic medications have been shown to impact gut microbiota (40, 64), which may play a role in their effects and side effects. Knowledge on the effect of atorvastatin on gut bacteria is scarce, but in vitro studies indicate that atorvastatin may inhibit the growth of some microbial species (65). Furthermore, bile acids may shape the gut microbiota community by promoting the growth of bile acid-metabolising bacteria and inhibiting the growth of other bile acid-sensitive bacteria (35, 36, 37). The healthy, young men participating here were medication-free and on a normal diet prior to enrolment and randomisation. We hypothesised that atorvastatin therapy would alter the gut microbiome either by directly inhibiting the growth of some species and favouring the growth of other species or indirectly through the alterations of the enterohepatic circulation of bile acids described above. Using a shotgun metagenomic approach, we obtained untargeted, high-resolution insights into the taxonomic composition and the functional potential of the gut microbiome of our participants following atorvastatin treatment. This analysis exhibited considerable inter-individual variation of even the most prevalent species, consistent with previous reporting (66). We found no difference when analysing microbiome community diversity, composition and functional metagenomic profiling before and after atorvastatin treatment and compared with placebo.

In conclusion, we show that 14 days of atorvastatin therapy in young, healthy men induces perturbations in gluco-metabolic hormones, amino acids and bile acids. We propose that the observed hyperglucagonaemia following atorvastatin treatment may constitute a link between statin treatment and the increased risk of incident diabetes and progression of diabetes seen in meta-analyses of randomised controlled trials and in observational studies.

Supplementary materials

Declaration of interest

MT: none; M-AM is an employee of Novo Nordisk; SSL: none; MCRDE is an employee of Novonesis; MBL: none, MLK: none; AB: none; JK is an employee of Novo Nordisk; OC-T: none; AT: none; BS: none; AD: none; BH: none; NJWA: none; JFR: none; JJH has, in the last three years, been an advisory/consultant for, been in the advisory board of and/or received research support from Eli Lilly, Novo Nordisk, Zealand Pharma, MSD Denmark, Structure Therapeutics, Scohia, HealthCap, Morgan Stanley, MEDACorp Inc., Arix Bioscience, Alphasights, Alcimed, Google Ventures Management, Guidepoint, Tema, Jefferies International Limited, Amgen, Sofinnova Partners, Thinks Insight & Strategy, AstraZeneca, Septerna; and co-founder of Antag Therapeutics and Villus (Bainan Biotech); TV has, in the last three years, served on scientific advisory panels, been part of speaker’s bureaus and served as a consultant to and/or received research support from Amgen, AstraZeneca, Boehringer Ingelheim, Eli Lilly, GSK, Mundipharma, MSD/Merck, Novo Nordisk, Sanofi and Sun Pharmaceuticals; A-ME: none; MOAS: none; DPS: none; FKK is advisor/consultant for, has been in the speaker’s bureau of and/or has received research support from 89bio, AstraZeneca, Boehringer Ingelheim, Cytoki Pharma, Eli Lilly, Gubra, Novo Nordisk, Merck Sharp & Dohme, Sanofi, Structure Therapeutics, Zealand Pharma and Zucara; FKK is a co-founder of and a minority shareholder in Antag Therapeutics, owns shares in Eli Lilly, Gubra, Novo Nordisk and Zealand Pharma and has been an employee of Novo Nordisk since 01 December 2023.

Funding

This work was supported by a grant from the Augustinus Foundation.

Author contribution statement

MT, TV, MOAS, DPS and FKK conceived the study; MT, MOAS, DPS and FKK designed the methodology; MT, M-AM and JK performed formal analysis; MT, M-AM, SSL, MCRDE, MLK, OCT, AT, BS, AD, BH, NJWA, JFR and JJH were involved in the investigation; MT wrote the original draft of the manuscript; MT, MBL, M-AM, A-ME, MOAS, DPS and FKK wrote, reviewed and edited the manuscript; MT and M-AM performed visualisation; AB, BS, TV, MOAS, DPS and FKK supervised the study; MT and FKK administered the project; and MT and FKK acquired funding. All authors approved the final version of the article.

Ethics statement

The trial was conducted in accordance with the Declaration of Helsinki, seventh revision, 2013. It was registered at the Danish Data Protection Agency and clinicaltrials.gov (NCT03018444). The protocol was approved by the Scientific Ethical Committee of the Capital Region of Denmark (reg. no. H-16034243). Written informed consent was obtained from all participants prior to any study-related activities.

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