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Keywords:

  • clinical trial;
  • diabetes;
  • endothelial function;
  • Hyperinsulinaemia the Outcome of its Metabolic Effects;
  • insulin;
  • low-grade inflammation;
  • metformin

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

Objectives.  The UK Prospective Diabetes Study (UKPDS) showed that treatment with metformin decreases macrovascular morbidity and mortality independent of glycaemic control. We hypothesized that metformin may achieve this by improving endothelial function and chronic, low-grade inflammation. Data on this issue are scarce and we therefore tested, in the setting of a randomized, placebo-controlled trial, whether metformin can affect endothelial function and low-grade inflammation.

Design.  The Hyperinsulinaemia the Outcome of its Metabolic Effects (HOME) trial is a double-blind trial, in which all patients were randomized to receive either metformin or placebo in addition to insulin therapy. At the beginning and the end of a 16-week treatment period fasting blood samples were drawn and a physical examination was carried out.

Setting.  The trial was conducted in the outpatient clinics of three nonacademic hospitals (Hoogeveen, Meppel and Coevorden; the Netherlands).

Subjects.  Patients were included if they were between 30 and 80 years of age; had received a diagnosis of diabetes after the age of 25; had never had an episode of ketoacidosis; and their blood glucose-lowering treatment previously consisted of oral agents but now only consisted of either insulin (n = 345) or insulin and metformin (n = 45). We excluded pregnant women and women trying to become pregnant, patients with a Cockroft-Gault-estimated creatinine clearance <50 mL min−1, or low plasma cholinesterase (reference value <3.5 units L−1), patients with congestive heart failure (New York Heart Association class III/IV), or patients with other serious medical or psychiatric disease. A total of 745 eligible patients were approached; 390 gave informed consent and were randomized (196 metformin, 194 placebo). About 353 patients completed 16 weeks of treatment (171 metformin, 182 placebo).

Main outcome measures.  The HOME trial was designed to study the metabolic and cardiovascular effects of metformin during a follow-up of 4 years. Presented here are the results of an interim analysis after 16 weeks of treatment.

Results.  When compared with placebo, metformin treatment was associated with an increase in urinary albumin excretion of 21% (−1 to +48; P = 0.06); a decrease in plasma von Willebrand factor of 6% (−10 to −2; P = 0.0007); a decrease in soluble vascular cell adhesion molecule-1 of 4% (−7 to −2; P = 0.0002); a decrease in soluble E-selectin of 6% (−10 to −2; P = 0.008); a decrease in tissue-type plasminogen activator of 16% (−20 to −12; P < 0.0001); and a decrease in plasminogen activator inhibitor-1 of 20% (−27 to −10; P = 0.0001). These changes could not be explained by metformin-associated changes in glycaemic control, body weight or insulin dose. Markers of inflammation, i.e. C-reactive protein and soluble intercellular adhesion molecule-1, did not change with metformin treatment.

Conclusions.  In patients with type 2 diabetes treated with insulin, metformin treatment was associated with improvement of endothelial function, which was largely unrelated to changes in glycaemic control, but not with improvement of chronic, low-grade inflammation.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

In type 2 diabetes mellitus, treatment with metformin has been associated with a decrease in macrovascular morbidity and mortality, which appears to be independent of the improvement in glycaemic control, as was demonstrated in the UK Prospective Diabetes Study (UKPDS) [1]. This observation suggests that metformin may affect the risk of atherothrombotic disease through mechanisms other than the lowering of blood glucose.

Two key features in the pathophysiology of atherothrombosis are dysfunction of the vascular endothelium and chronic, low-grade inflammation of the vascular wall [2]. Indeed, observational studies have found strong associations between markers of endothelial dysfunction and chronic, low-grade inflammation on the one hand, and increased risk of atherothrombotic disease on the other [3, 4].

These data raise the question of whether metformin can improve endothelial function and decrease inflammatory activity, and thereby decrease risk of atherothrombotic disease. There is some evidence from controlled studies that this may be the case [5–8], but most studies focused on markers of fibrinolysis only [6–8], which may or may not reflect endothelial function [9].

The randomized, placebo-controlled trial ‘Hyperinsulinaemia: the Outcome of its Metabolic Effects’ (HOME) was designed to investigate whether metformin, when compared with placebo but at a similar level of glycaemic control, decreases cardiovascular morbidity in patients with type 2 diabetes treated with insulin during a planned follow-up of 4 years. In view of the above considerations, we studied the effects of metformin on markers of endothelial function and low-grade inflammation in an interim analysis after 16 weeks of treatment. In addition, we explored biochemical mechanisms that might mediate metformin-associated improvements in endothelial function and inflammatory activity (if any).

Patients and procedures

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

The HOME trial was designed to study the metabolic and cardiovascular effects of metformin in insulin-treated type 2 diabetic patients [10]. We aimed to include 400 patients with type 2 diabetes mellitus between 30 and 80 years of age who had received a diagnosis of diabetes after the age of 25, who had never had an episode of ketoacidosis, and whose blood glucose-lowering treatment had previously consisted of oral agents but now only consisted of either insulin (n = 345) or insulin and metformin (n = 45). We excluded pregnant women and women trying to become pregnant, patients with a Cockroft–Gault-estimated creatinine clearance <50 mL min−1 [11], or low plasma cholinesterase (reference value <3.5 units L−1) [12] as a marker of liver failure. Patients with congestive heart failure (New York Heart Association class III/IV) or other serious medical or psychiatric disease were excluded as well.

All patients gave written informed consent. The medical ethical committees of the three participating hospitals approved the trial protocol. The trial has been and is being conducted in accordance with the Note for Guidance on Good Clinical Practice (CPMP/ICH/135/95) dated 17 July 1996 and in accordance with the Declaration of Helsinki (Edinburgh, 2000).

Study design

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

The HOME trial was conducted in the outpatient clinics of three nonacademic hospitals (Hoogeveen, Meppel and Coevorden). All patients were treated with insulin only for 12 weeks; four times daily (Actrapid preceding the three meals and Insulatard ante noctem; Novo Nordisk, Alphen a/d Rijn, the Netherlands) or twice daily [Mixtures of Actrapid (10–50%) and Insulatard (90–50%) preceding breakfast and dinner; Novo Nordisk]. After these 12 weeks, the 16-week short-term active treatment phase began at the start of which all subjects were randomly assigned to receive either metformin or placebo in addition to insulin therapy. All patients were numbered in order of study entry and were given trial medication with the same number. The boxes and tablets of metformin had a similar appearance. Each participant successively increased the dose from one to finally three tablets of 850 mg a day, if tolerated. The first tablet was taken at bedtime, the second at breakfast and the third at dinner. The treatment goals were fasting plasma glucose levels between 4 and 7 mmol L−1 and postprandial glucose levels between 4 and 10 mmol L−1. At the beginning and the end of this 16-week short-term active treatment phase, fasting blood samples were drawn, a physical examination was carried out, and a complete medical history was taken. We have previously reported results on glycaemic control, weight gain, and insulin dose [10], vitamin B12, folate, and homocysteine [13], and ambulatory blood pressure (M. G. Wulffelé, A. Kooy, P. Lehert, D. Bets, A. J. M. Donker, C. D. A. Stehouwer, personal communication).

Laboratory investigations

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

The laboratories of the three hospitals used standard analytical methods with the same reference values for laboratory measurements on fasting plasma glucose, haemoglobin A1c (HbA1c), fasting lipid concentrations and urinary albumin excretion. Plasma glucose levels were determined using an automated glucose oxidase method (Hitachi 917; Roche, Basel, Switzerland) in Hoogeveen and Meppel. HbA1c (normal value 4.0–6.0%) was measured by high-performance liquid chromatography in Hoogeveen and by an immunoturbidimetric method (Unimate; Roche) in Meppel. Fasting lipid concentrations were assessed by standard methods. The Coevorden hospital used dry chemistry for all above-mentioned laboratory measurements (Orthoclinical Diagnostics; Johnson and Johnson, Rochester, NY, USA). Plasma low-density lipoprotein (LDL) cholesterol was directly determined by the N-geneous TM assay (Genzyme, Cambridge, MA, USA). Urinary albumin excretion (at baseline the mean of three fresh morning urine collections) was measured by immunoturbidimetry (Roche Diagnostics, Basel, Switzerland) in Meppel and Hoogeveen and by means of nephelometry (BN ProSpec, Dade Behring, Marburg, Germany) in Coevorden. Urinary albumin excretion was expressed as the albumin-to-creatinine ratio. Method comparison according to Passing and Bablok [14, 15] showed no significant deviation between these methods. In addition, by means of a randomized block test, no significant difference in HbA1c values between the three hospitals was found.

We measured plasma von Willebrand factor (vWf) antigen and C-reactive protein (CRP) with highly sensitive in-house sandwich enzyme immunoassays. Rabbit antihuman vWf or CRP immunoglobulins were used as catching antibodies; peroxidase-conjugated rabbit antihuman vWf or CRP immunoglobulins were used as detecting antibodies (Dako, Copenhagen, Denmark). o-Phenylenediamine (Sigma Chemical Co., St Louis, MO, USA) acted as substrate for both plasma vWf and CRP antigen. Levels of vWf are expressed as percentage of antigen levels in normal pooled plasma, which is defined as 100%. The intra- and interassay coefficients of variation were 2.0% and 5.7% for plasma vWf antigen and 3.9% and 8.7% for CRP, respectively.

We measured plasma levels of soluble (s) vascular cell adhesion molecule-1 (VCAM-1; Diaclone, Besançon, France), soluble intercellular adhesion molecule-1 (ICAM-1; Diaclone), soluble E-selectin (sE-selectin; Bender MedSystems, Vienna, Austria), tissue-type plasminogen activator (t-PA) antigen (Biopool International, Umeå, Sweden), and plasminogen activator inhibitor-1 (PAI-1) antigen (Innogenetics, Gent, Belgium) in duplicate by use of commercially available enzyme-linked immunosorbent assay (ELISA) kits. The intra- and interassay coefficients of variation were 4.4% and 4.6% for sVCAM-1; 4.0% and 7.4% for sICAM-1; 3.1% and 11.9% for sE-selectin; 2.8% and 7.5% for tPA; and 2.8% and 8.2% for PAI-1, respectively.

The LDL particle size was measured by high-performance gel-filtration chromatography (HPGC) with fluorescence detection after postcolumn labelling with parinaric acid, a fluorescent lipid probe [16, 17]. Both intra- and interassay coefficients of variation were <0.25%. Samples used for the determination of vWf, CRP, sVCAM-1, sICAM-1, t-PA, PAI-1 and LDL particle size were stored at −80 °C until subsequent analysis.

We considered urinary albumin excretion and plasma levels of vWf, sVCAM-1, sE-selectin, t-PA and PAI-1 as markers of endothelial function [18–20] and plasma CRP and sICAM-1 as markers of inflammatory activity [20–22].

Statistical analyses

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

We included only subjects with complete data sets (n = 313). All data with a skewed distribution were log-transformed before analysis.

The end-point of interest was the percentage change of each variable from baseline, and the differences in these changes between the metformin and the placebo group. The differences between the metformin and placebo group were tested by means of a Student's t-test on log-transformed values. As log-values are not directly interpretable, the antilogs are reported instead. In case of log-transformed values, data are given as geometric mean (95% CI).

We used multiple linear regression analysis to investigate whether metformin-associated improvements in markers of endothelial function and markers of inflammation, if any, were independent of changes in HbA1c, fasting or postprandial plasma glucose, insulin dose, body mass index (BMI) and LDL cholesterol concentration or particle size. A P-value <0.05 was considered statistically significant.

Patients

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

We screened the medical files of all three participating outpatient clinics and identified 745 eligible patients (Fig. 1). They were all invited to enrol in the trial and 390 subjects gave written informed consent. The subjects were subsequently randomized to receive either metformin (196 subjects) or placebo (194 subjects). Of these 390 patients, 37 dropped out; 25 in the metformin group and 12 in the placebo group. Two patients never took their medication (placebo one, metformin one); nine patients withdrew their consent (placebo five, metformin four), and 26 experienced adverse effects (placebo six, metformin 20). Of these 26 patients, 11 experienced diarrhoea (placebo two, metformin nine); five encountered flatulence (placebo one, metformin four); four suffered fatigue (placebo one, metformin three); one experienced pruritus (metformin); one had headaches (metformin); one had pyrosis (placebo); one had nausea (metformin), one had a myocardial infarction (placebo), and one patient died suddenly (metformin).

image

Figure 1. Trial profile.

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Table 1 shows the baseline characteristics at the start of the short-term active treatment phase. Patients randomized to receive metformin were slightly older than patients randomized to receive placebo (63.2 ± 9.8 vs. 58.9 ± 11.1 years), but other characteristics were comparable between the two groups. There were no differences in the use of acetyl salicylic acid (ASA) or type of antihypertensive medication used (Table 1). The actual mean dose in the metformin-treated group was 2163 mg. Each patient maintained his/her maximally tolerated daily dose (1, 2 or 3 tablets of 850 mg) throughout the 16 weeks of treatment.

Table 1.  Baseline characteristics of the ‘per protocol population’ of 313 patients
 Placebo (n = 163)Metformin (n = 150)
  1. Data are given as mean (SD) or number (%).

  2. HDL, high-density lipoprotein; LDL, low-density lipoprotein; HbA1c, haemoglobin A1c.

Demography
 Men/women  84/79  66/84
 Age (years)  59 (11)  63 (9)
 Currently smoking, n (%)  50 (31)  31 (21)
 Duration of diabetes (years)  12 (8)  14 (8)
 Insulin treatment (years)    6 (6)    6 (7)
Diabetic complications
 Cardiovascular, n (%)  53 (29)  59 (35)
 Retinal coagulation and (or) cataract extraction, n (%)  25 (14)  35 (22)
 Amputation, n (%)    3 (2)    4 (2)
 Paraesthesias, n (%)  79 (43)  83 (49)
Concomitant medication
 Acetyl salicylic acid, n (%)  25 (15)  29 (19)
 Lipid-lowering drugs, n (%)  36 (20)  34 (20)
 Blood pressure-lowering drugs, n (%)  73 (40)  88 (51)
   Enalapril  27 (17)  25 (17)
   Losartan    3 (2)    3 (2)
   Lercanidipine    1 (1)    2 (1)
   Thiazide    9 (6)  10 (7)
Clinical features
 Body mass index (kg m−2)  29.5 (4.6)  29.9 (5.2)
 Weight (kg)  86.2 (14.6)  85.6 (15.7)
 Waist-to-hip ratio
   Men    1.03 (0.09)    1.01 (0.07)
   Women    0.93 (0.09)    0.93 (0.09)
 Systolic blood pressure (mmHg)159 (25)160 (26)
 Diastolic blood pressure (mmHg)  85 (11)  86 (12)
 Daily dose of insulin (IU day−1)  63 (26)  64 (30)
Laboratory variables
 Fasting plasma glucose (mmol L−1)  10.3 (3.2)  10.0 (3.0)
 Total cholesterol (mmol L−1)    5.5 (1.3)    5.6 (1.1)
 HDL cholesterol (mmol L−1)    1.3 (0.4)    1.3 (0.4)
 LDL cholesterol (mmol L−1)    3.4 (1.0)    3.6 (1.0)
 Triglycerides (mmol L−1)    1.9 (1.5)    1.7 (1.1)
 HbA1c (%)    7.8 (1.2)    7.8 (1.2)

Markers of endothelial function

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

When compared with placebo, metformin treatment was associated with an increase in urinary albumin excretion of 21% (-1 to +48; P = 0.06); a decrease in vWf of 6% (−10 to −2; P = 0.0007); a decrease in sVCAM-1 of 4% (−7 to −2; P = 0.0002); a decrease in sE-selectin of 6% (−10 to −2; P =0.008); a decrease in t-PA of 16% (−20 to −12; P < 0.0001); and a decrease in PAI-1 of 20% (−27 to −10; P = 0.0001) (Table 2).

Table 2.  Markers of endothelial function and of inflammation at baseline and after 16 weeks of treatment with placebo or metformin
 Baseline (t0)16 weeks (t1)Change (%)P-value (metformin versus placebo)
PlaceboMetforminPlaceboMetformin Placebo (t1 versus t0) Metformin (t1 versus t0)Metformin versus placebo
  1. Data at baseline and follow-up are presented as mean with 95% CI, or, when log-transformed, as geometric mean with 95% CI. Change is expressed as the mean percentage of change accompanied with a 95% CI. Reported P-values are the result of Student's t-tests on differences in change between the metformin and the placebo group.

  2. vWf, von Willebrand factor; sVCAM-1, soluble vascular cell adhesion molecule-1; t-PA, tissue-type plasminogen activator; PAI-1, plasminogen activator inhibitor-1; CRP, C-reactive protein; sICAM-1, soluble intercellular adhesion molecule-1; sE-selectin, soluble E-selectin; HbA1c, haemoglobin A1c; BMI, body mass index; LDL, low-density lipoprotein; CI, confidence interval.

Markers of endothelial function
 Urinary albumin excretion (mg mmol−1)1.2 (0.9–1.5)1.0 (0.8–1.3)1.2 (1.0–1.6)1.2 (1.0–1.6)+4 (−11 to +20)+26 (+11 to +43)+21 (−1 to +48)0.06
 vWf (%)136 (129–143)143 (136–151)137 (130–143)135 (128–142)0 (−3 to +4) −6 (−9 to −3) −6 (−10 to −2)0.0007
 sVCAM-1 (ng mL−1)879 (850–909)919 (885–954)883 (852–915)878 (849–907)0 (−2 to +2) −4 (−7 to −2) −4 (−7 to −2)0.0002
 sE-selectin (ng mL−1)47.3 (44.1–50.8)51.0 (47.9–54.2)47.9 (44.8–51.3)48.6 (45.6–51.8)+1 (−2 to +5) −5 (−7 to −2) −6 (−10 to −2)0.008
 t-PA (ng mL−1)11.8 (11.1–12.6)12.7 (11.9–13.4)12.0 (11.3–12.7)10.7 (10.0–11.4)+1 (−2 to +5) −15 (−18 to −12) −16 (−20 to −12)<0.0001
 PAI-1 (ng mL−1)95.0 (84.8–106)94.5 (83.6–107)98.8 (87.9–111)79.4 (69.9–90.2)+4 (−3 to +12) −16 (−12 to −9) −20 (−27 to −10)0.0001
Markers of inflammation
 CRP (mg L−1)3.27 (2.80–3.81)3.26 (2.80–3.78)3.34 (2.89–3.86)3.24 (2.80–3.75)+2 (−8 to +14) −1 (−10 to +10) −3 (−16 to +12)0.7
 sICAM-1 (ng mL−1)604 (580–628)614 (590–639)601 (579–623)602 (577–628)0 (−3 to +2) −2 (−4 to 0) −2 (−4 to +1)0.3
Markers of glycaemia and other variables
 HbA1c (% Hb)7.8 (7.6–8.0)7.8 (7.6–7.9)7.5 (7.4–7.7)6.9 (6.7–7.0) −3 (−5 to −2) −11 (−13 to −10) −8 (−11 to −6)<0.0001
 Fasting plasma glucose (mmol L−1)9.2 (8.9–9.5)9.2 (8.9–9.5)8.6 (8.4–8.8)7.6 (7.4–7.8) −7 (−9 to −5) −17 (−20 to −15) −11 (−14 to −8)<0.0001
 Postprandial plasma glucose (mmol L−1)10.0 (9.7–10.3)10.0 (9.7–10.4)8.6 (8.4–8.9)7.8 (7.6–8.0) −14 (−16 to −11) −22 (−25 to −19) −10 (−19 to −6)<0.0001
 Insulin dose (U kg−1)0.7 (0.2–1.2)0.7 (0.2–1.3)0.8 (0.2–1.3)0.7 (0.0–1.4)+8 (−20 to +36)0 (−23 to +33) −8 (−11 to −4)<0.0001
 BMI (kg m−2)29.6 (20.5–38.7)29.6 (19.8–39.5)30.0 (19.4–40.7)29.5 (19.5–39.4)+2 (−12 to +15) −1 (−7 to +5) −2 (−3 to −1)0.0006
 LDL cholesterol (mmol L−1)3.4 (3.3–3.6)3.6 (3.4–3.7)3.4 (3.3–3.6)3.4 (3.2–3.5)0 (−2 to +2) −6 (−8 to −3) −6 (−9 to −3)0.0003
 LDL particle size (nm)21.60 (21.52–21.68)21.67 (21.58–21.75)21.59 (21.52–21.67)21.69 (21.61–21.77)0 (0–0)0 (0–0)0 (0–0)0.4
 Triglycerides (mmol L−1)1.5 (1.3–1.6)1.4 (1.2–1.5)1.5 (1.4–1.7)1.4 (1.2–1.5)+3 (−4 to +10)0 (−6 to +6) −3 (−12 to +7)0.6

Markers of glycaemia and other variables

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

When compared with placebo, metformin treatment was associated with a decrease in HbA1c of 8% (−11 to −6; P < 0.0001); a decrease in fasting plasma glucose of 11% (−15 to −8; P < 0.0001); a decrease in postprandial plasma glucose of 9% (−13 to −5; P < 0.0001); a decrease in insulin dose of 8% (−11 to −4; P < 0.0001); a decrease in BMI of 2% (−3 to −1; P = 0.0006); and a decrease in LDL cholesterol concentration of 6% (−9 to −3; P = 0.0003). LDL particle size and triglyceride concentrations did not change significantly during metformin or placebo treatment (Table 2).

Additional analyses

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

First, we carried out adjustments for age, sex, menopausality, blood pressure, use of ASA and antihypertensive medication, and for baseline values of the markers for endothelial function and inflammation to rule out that a nonsignificant imbalance in these variables at baseline amongst the two treatment groups might has affected the outcome. These adjustments did not change any of the results (data not shown). Secondly, to investigate whether metformin-associated changes in markers of endothelial function and inflammation were mediated by metformin-associated changes in other variables (i.e. HbA1c, fasting or postprandial plasma glucose, insulin dose, BMI, LDL cholesterol concentration or particle size and triglyceride concentration) we adjusted for changes in these variables in the analyses. This had no effect on the changes in vWf, sVCAM-1, t-PA, and PAI-1, CRP or sICAM-1. However, the difference in sE-selectin between the metformin and placebo group was in part (about 25%) dependent on the difference in fasting plasma glucose and triglyceride between the groups. In addition, the increase in urinary albumin excretion in the metformin group became statistically significant after adjustment for HbA1c (+32%, +7 to +63 vs. placebo; P = 0.01), which decreased under metformin treatment (−8%, −11 to −6 vs. placebo; P < 0.0001), and this decrease in HbA1c was itself associated with a decrease in urinary albumin excretion (1.4%, 1.0–1.9 per 1% HbA1c decrease).

Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

We found that, when compared with placebo, 16 weeks of metformin treatment in patients with type 2 diabetes intensively treated with insulin was associated with significant improvement of endothelial function but not of chronic, low-grade inflammation.

Type 2 diabetes is a state of generalized endothelial dysfunction, i.e. there is impairment of many endothelial functions, such as regulation of vasomotor tone, leucocyte adhesion, haemostasis and fibrinolysis, in many vascular beds [18]. Endothelial dysfunction in type 2 diabetes tends to be progressive and is strongly associated with cardiovascular disease risk [3]. We found that metformin treatment was associated with decreases in the plasma levels of vWf, sVCAM-1, sE-selectin, t-PA and PAI-1, i.e. with improvement of the endothelial regulation of haemostasis (vWf), leucocyte adhesion (sE-selectin and sVCAM-1) and fibrinolysis (t-PA and PAI-1). For vWf, sE-selectin, t-PA and PAI-1, these findings are in accordance with previous experience in diabetic and nondiabetic individuals [6–8, 23–25]. Interestingly and importantly, changes in the plasma levels of these markers were independent of metformin-associated favourable changes in body weight, glycaemic control, insulin dose, and LDL cholesterol concentration, and they were also independent of LDL particle size, and triglyceride concentrations. The only exception was the change in plasma sE-selectin, which was in part explained by the metformin-associated improvement in glycaemic control and by triglyceride levels, in accordance with previous data on the role of glucose and lipids in the regulation of E-selectin synthesis [26–28]. In addition, changes in the plasma levels of the markers could not be attributed to the influence of the other variables studied. LDL particle size did not change upon treatment and therefore, the decrease in LDL cholesterol concentration could not in any part be explained by decreasing LDL particle size, a phenomenon which has been associated with an increased cardiovascular risk [29]. Smoking habits did not differ between the two groups at baseline with respect to the number of cigarettes smoked per day (M 2.1, P 3.3), or with respect to the number of patients who quit smoking in the 16-week follow-up period. The number of smokers was somewhat more unbalanced between the groups [M 31 (21%); P 50 (31%)]. This baseline difference, however, has not led to any difference in vascular function at baseline, as measured by the markers of endothelial function and inflammation. No differences in blood pressure existed at baseline or after 16 weeks of treatment with metformin, as published previously (M. G. Wulffelé, A. Kooy, P. Lehert, D. Bets, A. J. M. Donker, C. D. A. Stehouwer, personal communication). No difference in alcoholic consumption existed between the two groups. Data on adherence to lifestyle were not available. Taken together, these findings raise the possibility that improvement of endothelial function by metformin may represent a largely glucose-independent pathway through which metformin decreases risk of cardiovascular disease in type 2 diabetes [1].

An important assumption in this reasoning is that plasma levels of these markers are valid indicators of endothelial function. This, in turn, requires that endothelial cells are the major source of the plasma concentrations of these proteins, and that protein concentration is determined by synthesis rather than by clearance. The validity of these assumptions is uncertain [18]. Only sE-selectin and t-PA are synthesized exclusively by endothelial cells. However, t-PA in plasma binds to PAI-1, and t-PA concentrations may mainly reflect the concentration of PAI-1, which is synthesized not only by endothelial cells, but also by hepatocytes and adipocytes. In addition, there is virtually no information on the regulation of the clearance of these proteins in type 2 diabetes, except for vWf, for which there is indirect evidence that its plasma concentration, in type 2 diabetes, is determined by synthesis rather than clearance [30].

Unexpectedly, urinary albumin excretion tended to increase slightly during short-term treatment with metformin, an increase that was statistically significant after adjustment for changes in HbA1c. Notably, a decrease in HbA1c, as expected, was itself associated with a decrease of urinary albumin excretion. Taken together, these results suggest that metformin can decrease urinary albumin excretion by improving glycaemic control, whilst at the same time it can increase urinary albumin excretion through other mechanisms. Previous studies of the effect of metformin on urinary albumin excretion showed either no effect [31, 32] or a decrease [6, 33]. Nevertheless, our results are unexpected, and we cannot exclude that they represent a chance finding. In addition, even if the findings are valid, their interpretation is unclear. Small increases in urinary albumin excretion, i.e. microalbuminuria, are strongly associated with endothelial dysfunction and have been postulated to reflect increased endothelial permeability to macromolecules [34]. This concept may not hold under all circumstances, however, because urinary albumin excretion depends on glomerular albumin permeation (i.e. pressure, permeability and surface area) and tubular reabsorption. The discrepancy between the effects of metformin on urinary albumin excretion (unfavourable) and those on the plasma markers of endothelial function (favourable) raises the possibility that the effect of metformin on urinary albumin excretion may be unrelated to endothelial function. These possibilities require further investigation.

We found no effect of short-term treatment with metformin on markers of low-grade inflammation, i.e. sICAM-1 and CRP. To the best of our knowledge, no placebo-controlled trials designed to study the effect of metformin on sICAM-1 or CRP in diabetes have been reported. However, a study in women with polycystic ovary syndrome (PCOS) reported a decrease in CRP after metformin treatment [35]. The differences between diabetic and PCOS patients require further investigation.

We showed that the effects of metformin on endothelial function were mostly unrelated to decreases in hyperglycaemia, insulin dose and BMI, suggesting that metformin may have direct effects on the endothelium [36–38]. However, we cannot exclude that metformin improves endothelial function by decreasing advanced glycation end-product levels [39–41], by altering the secretion of adipocyte-derived mediators (such as free fatty acids, leptin, resistin and adiponectin) [42–48], by decreasing inflammatory activity in ways not reflected by CRP and sICAM-1 [49, 50], and (or) by improving insulin sensitivity, of which a change in insulin dose may be an insufficiently accurate marker. These possibilities require further study.

We conclude that, in patients with type 2 diabetes treated with insulin, 16 weeks of metformin treatment, when compared with placebo, was associated with improvements in plasma markers of endothelial function, which were mostly unrelated to changes in HbA1c, fasting or postprandial plasma glucose, insulin dose, BMI, LDL cholesterol and triglyceride concentrations. Metformin may thus have specific effects on endothelial function, which may explain, in part, why metformin appears to be associated with a decreased risk of cardiovascular disease in type 2 diabetes. This hypothesis requires further testing.

Conflict of interest statement

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

Adriaan Kooy, as principal investigator, received grants to support the HOME-study from Byk, Lifescan, Merck-Santé, Merck, Sharpe & Dohme and Novo Nordisk. Philippe Lehert is an occasional consultant for Merck-Santé. Daniel Bets is an employer of Merck B.V., responsible as trial monitor for the proper conduct of the study. For the other authors no conflict of interest exists.

Acknowledgements

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References

This part of the HOME-trial was supported by grants from Byk, Lifescan, E. Merck/Lipha, Merck, Sharpe & Dohme and Novo Nordisk. Authors thank the study nurses Liesbeth Breedland and Els van Driesum for their dedication to the concerns of the patients and the quality of the treatments, Jan van der Kolk for his technical assistance and validation of the laboratory techniques, Gerard de Groot, Rob Hoorn, and Erk Pieterse for their hospitality at their laboratories, and all other members of the HOME-study group for their contribution to the HOME-trial.

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and methods
  5. Patients and procedures
  6. Study design
  7. Laboratory investigations
  8. Statistical analyses
  9. Results
  10. Patients
  11. Markers of endothelial function
  12. Markers of inflammation
  13. Markers of glycaemia and other variables
  14. Additional analyses
  15. Discussion
  16. Conflict of interest statement
  17. Acknowledgements
  18. References
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