2.1 Diagnosing type 2 diabetes mellitus

In our studies the diagnosis of type 2 diabetes mellitus was based on both clinical and biochemical evaluation of patients as suggested by Hother-Nielsen et al (34). Patients were classified as having type 2 diabetes mellitus according to the following criteria: 1) onset of diabetes after the age of 40; 2) a glucagon-stimulated serum C-peptide value ≥ 600 pmol/l (35-37).

2.2 Microalbuminuria

As mentioned previously microalbuminuria defined as a persistently elevated UAER in the range 30 to 300 mg per 24 hour is a risk factor for both micro- and macrovascular late diabetic complications (38). Therefore, we chose to examine microalbuminuric type 2 diabetic patients in order to have a well defined study cohort. However, It should be mentioned that microalbuminuria in patients with type 2 diabetes may not be as homogeneous as expected. First, since microalbuminuria at baseline was determined while a large amount of patients were already treated with antihypertensive medication, we may have included some patients with "masked" nephropathy since, as it will be discussed in later sections, antihypertensive treatment lowers UAER. Second, renal structural lesions in patients with type 2 diabetes and microalbuminuria may be quite heterogeneous as suggested by Fioretto et al (39). In this study kidney biopsies from 34 microalbuminuric, unselected type 2 diabetic patients were examined. Thirty percent of patients had normal or near normal renal structure, 30% had "typical" lesions characteristic for diabetic nephropathy (glomerular, tubulo-interstitial and arteriolar changes occurring in parallel) while 40% had "atypical" patterns of injury, with absent or only mild diabetic glomerular changes and concomitant disproportionately severe renal structural changes, which included tubular atrophy, tubular basement membrane thickening, interstitial fibrosis, advanced glomerular hyalinosis and global glomerular sclerosis. While none of the patients in the latter group had any definable non-diabetic renal disease, some of these lesions may be caused by renal ischaemia as a result of atherosclerotic renal artery stenosis or cholesterol microembolism (40).

As it will be described in later sections microalbuminuria is a strong risk factor for both cardiovascular disease as well as late-diabetic microvscular complications, thus the inclusion criterion of microalbuminuria in the Steno-2 ensured a high risk population for these complications.

2.3 Ethnic origin

Large variations in the prevalence of type 2 diabetes and its complications have been described among patients of different ethnic origin (41-43). To minimise heterogeneity by the potential confounding effect of race we therefore in the present studies chose only to include patients who were Danish Caucasians by self-report.

2.4 Composition and sampling of the study population

The overall purpose of the patient selection was to study a group of type 2 diabetic patients with well defined characteristics as mentioned above. All patients were recruited from Steno Diabetes Center. For the multifactorial intervention study (44-47) and epidemiological studies (48; 49) all type 2 diabetic patients aged 40 to 65 years who during 1992 had a UAER of 30 to 300 mg per 24 hour in a single urine sample were eligible (n=315). Thirty-seven patients refused to participate, 104 patients did not fulfil our criteria for microalbuminuria, 9 patients had a stimulated serum C-peptide level below 600 pmol/l and 5 were excluded of other causes giving a total of 160 patients who with concealed randomisation were divided into two treatment groups in an open, parallel study comparing intensified to conventional multifactorial intervention. Two other studies were carried out as randomised, double-blind, cross-over trials (50; 51). In the vitamin trial (50) 37 consecutive type 2 diabetic patients with microalbuminuria according to definition were eligible. Since treatment with ACE inhibitors were withdrawn eight weeks before treatment with vitamin C and E or placebo patients with prior myocardial infarction or congestive heart failure were excluded from the study (n=4) and three refused to participate giving a total of 30 patients who were randomised. Finally, one patient withdrew during treatment pause, but before active/placebo treatment was initiated. In the aspirin trial treatment with ACE inhibitors was also stopped eight weeks before the first treatment phase and similar exclusion criteria as in the vitamin trial were applied (51). Furthermore, a history of stroke or transitory cerebral ischaemia, peptic ulcer disease, allergy to aspirin and use of cyclooxygenase inhibitors were exclusion criteria. Thirty-one patients out of a total of 43 eligible patients were randomised (two patients refused, four had prior myocardial infarction, two had prior cerebral thrombosis, three used cyclooxygenase inhibitors, and one had a gastrointestinal ulcer). All randomised patients completed this study.

4.1 Kidney function

4.1.1 Glomerular filtration rate (GFR)

GFR was estimated in the supine position from plasma clearance following a single bolus injection of 3.7 MBq 51 Cr-labelled edetic acid in the morning by determining the radioactivity in venous blood samples taken from the other arm 180, 200, 220 and 240 minutes after the injection with appropriate corrections and standardisation for the patient's surface area (57-59).

When progression in any chronic kidney disease is evaluated development of ESDR is the ultimate endpoint. Since it takes several years to reach ESRD clinical trials in progressive kidney disease often requires other endpoints. The rate of decline in GFR has been approved as an endpoint by the Food and Drug Administration (USA). The rate of decline in GFR was only calculated for patients completing follow-up (44; 46) or was calculated as the difference between first and last GFR determination in all participating patients (51).

4.1.2 Urinary albumin excretion rate

Due to large day to day variation in UAER (60) three 24-hour urinary collections were performed at each designated time point in all our studies. Indeed six 24-hour collections were used to confirm microalbuminuria at baseline in two studies (44; 46). Timed urinary collections were used as it is widely accepted as the most accurate method for determination of UAER (61). The urinary albumin concentration was determined by an immunoassay method (62). Normoalbuminuria was defined as UAER < 30 mg per 24 hour, microalbuminuria as UAER 30-300 mg per 24 hour, and macroalbuminuria as UAER > 300 mg per 24 hour as defined at a consensus conference (63). The fractional clearance of albumin was calculated by dividing the clearance of albumin (calculated as UV/P, where U is urine albumin concentration (mg/l), V is urine flow (l/24 h), and P is plasma albumin concentration (g/l)) with the simultaneous measured GFR to correct albumin excretion for changes in plasma albumin concentration and in GFR (51).

4.2 Endothelial dysfunction

Endothelial function was evaluated by determining the transcapillary escape rate of albumin (TER alb ) in one study (51) and with measurement of the serum concentration of von Willebrand factor (vWF) after an overnight fast (48).

4.2.1 Transcapillary escape rate (TER alb )

TER alb is defined as the fraction of the intravascular mass of albumin that passes to the extravascular space per unit of time (percent per hour). It is determined as the rate constant of the practically monoexponential decrease in plasma radioactivity over the first 60 minutes following injection of tracer albumin as calculated by the least squares method as described (64).

4.2.2 Von Willebrand factor

Von Willebrand factor is a high molecular weight glycoprotein synthesised mainly by the endothelial cells and acts as a non-specific marker of endothelial dysfunction (65; 66). The plasma concentration of vWF was measured by microenzyme linked immunoadsorbent assay as described by Ingerslev (67). A close agreement between plasma and serum levels of vWF has been described (68).

4.3 Laboratory assays

4.3.1 Glycaemic regulation

Glycosylated haemoglobin A 1c was determined by ion-exchange high-performance liquid chromatography (HPLC) (Bio-Rad VARIANT, California, USA) and the non-diabetic reference range in our laboratory was 4.1-6.4%. Blood glucose was measured by a glucose oxidase method.

4.3.2 Lipid profiles

Venous blood samples were drawn after a 12 hour fast. Serum total cholesterol and serum high density lipoprotein (HDL)-cholesterol were measured by chromatography and serum triglycerides were measured by colorimetry. Serum low density lipoprotein (LDL)-cholesterol was calculated by the Friedewald formula (69) in patients with a serum triglyceride concentration lower than 5 mmol/l.

4.3.3 Pancreatic β -cell function

Serum C-peptide concentration was measured by radioimmunoassay (RIA) (70) in the fasting state and 6 minutes after intravenous injection of 1 mg glucagons to measure residual β -cell function (35).

4.3.4 Vitamin E and C

Plasma α -tocopherol, ascorbic acid and its metabolite dihydro-ascorbic acid were measured by HPLC (71; 72).

4.3.5 Plasma NT-proBrain Natriuretic Peptide(NT-proBNP)

After the patients had been at rest for at least 20 minutes in the supine position, blood samples (EDTA plasma) for analysis of plasma NT-proBNP were collected, centrifuged and plasma stored at -80°C until analysis. Plasma concentrations of NT-proBNP were measured by a sandwich immunoassay on an Elecsys 2010 (Roche Diagnostics, Germany). The analytical range extends from 5 to 35 000 pg/ml, and the total coefficient of variation is < 0.061 in pooled human plasma samples (73). To convert from pg/ml to pmol/l multiply by 0.118.

4.4 Arterial blood pressure

Arterial blood pressure was measured with a Hawksley random zero sphygmomanometer (Hawksley & Sons Ltd, Lancing, Sussex, UK) and by the use of an automatic, oscillometric manometer (Takeda Medical UA-751, Tokyo, Japan).

Arterial blood pressure was after an overnight fast measured twice on both arms in the supine position with the random zero device after 20 minutes rest by a laboratory technician unaware of both treatment allocation and actual treatment and the average of these four measurements was used. Cuff size 25 × 12 cm was used if the upper arm circumference was below 35 cm, and cuff size 30 × 15 cm was used if upper arm circumference was equal to or above 35 cm. Diastolic blood pressure was recorded at the disappearance of Kortokoff sounds (phase 5). Patients did not take any medication before blood pressure measurements. The Hawksley random zero sphygmomanometer was used to exclude bias in the readings as compared to a simple mercury manometer. Yet, the Hawksley apparatus has been criticised for being inaccurate, especially in the measurement of systolic blood pressure, where an underestimation of 2 to 4 mm Hg was found (74; 75).

The automated Takeda UA-751 manometer was used for determination of the reference arterial blood pressure in the systolic arm-toe gradient and for the blood pressure measurements for orthostatic hypotension. An appropriate cuff size as mentioned above was used. The Takeda UA-751 gives difference in systolic blood pressure of -0.11 +/- 5.6 mm Hg (mean +/- SD) and in diastolic blood pressure of 0.31 +/- 5.5 mm Hg as compared to a mercury sphygmomanometer (76).

4.5 Retinopathy

Two mydriatic 60-degree fundus photographs were taken on 35 mm colour transparency film one covering the macula-temporal part of the retina and one covering the optic disc and nasal part of the retina. The photographs were graded according to the EURODIAB six-level grading scale (77) by two independent graders masked for treatment allocation (44; 46). Any presence of maculopathy was determined by evaluation of a stereo-set of photographs of the macula region and data were drawn from the files of the eye clinic at Steno Diabetes Center. The evaluation of any maculopathy was masked for treatment allocation and done by graders independent from our studies at the Steno Diabetes Center. Visual acuity was measured with dilated pupils by a Nikon NR-7000 autorefractor with a cut off upper limit of 1.0 refracted visual acuity. Blindness was defined by WHO criteria as a visual acuity equal to or less than 0.1.

4.6 Diabetic neuropathy

4.6.1 Autonomic nervous function

The beat-to-beat variation is a simple bedside test that mainly evaluates cardiac vagal function. The interpretation of the test results as originally proposed by Hilsted et al (78) (abolished < 4 beats/min, impaired 5-15 beats/min and normal > 15 beats/min) has been challenged, since it has been shown that beat-to-beat variation decreases with older age (79-81). Indeed the overestimation of the prevalence of impaired cardiac vagal function in middle-aged type 2 diabetic patients by the use of normal values derived from young healthy subjects by applying the original criteria was obvious in a cross-sectional study by Nielsen et al (33) since the prevalence of impaired and abolished beat-to-beat variation was very high in the non-diabetic control group. As a consequence we used values obtained from this non-diabetic control group as normal values (abolished < 4 beats/min, impaired 4-6 beats/min and normal > 6 beats/min). We used 3 lead electrocardiogram (ECG) monitoring with a paper velocity of 25 mm/second. Beat-to-beat variation was calculated as the difference between maximal and minimal heart rate during in- and expiratory phase. The mean value obtained from 5 in- and expiratory cycles was used.

Autonomic sympathetic nervous function was evaluated by orthostatic blood pressure test. Arterial blood pressure was measured twice in the right arm after 30 minutes rest in the supine position. Systolic blood pressure was recorded at 0.5, 1.5, 3, 5 and 7 minutes after rising to an upright position. Orthostatic hypotension was diagnosed if the maximal fall in systolic blood pressure exceeded 25 mm Hg in any of the measurements (82).

4.6.2 Peripheral nervous function

Measurement of the vibration threshold with the use of a biothesiometer was used to evaluate peripheral neuropathy. We used an age-adjusted scale as described by Bloom et al (83). Testing was standardised so that the tactor was held in firm contact but with minimal pressure against the skin. The plantar aspect of the great toe opposite the nail bed was used. Although stockings and thin socks did not alter the thresholds in the reference population (83) measurements in our studies were obtained on the bare foot. In case of amputation of the first toe, measurements were done in the adjacent toe.

4.6.3 Are the methods used in the evaluation of neuropathy reliable?

Diabetic polyneuropathy presents with a wide range of symptoms reflecting the broad range of nerve fibre types involved. According to the American Diabetes Association and the Rochester Diabetic Neuropathy Study evaluations for diagnosis and staging of diabetic polyneuropathy should include assessment of a) neuropathic symptoms, b) neuropathic deficits, c) nerve conduction, d) quantitative sensory examination, and e) quantitative autonomic examination (84; 85). The protocol applied in the Steno-2 study does not fulfil all of these criteria. Whereas the methods used for measurement of autonomic nerve function (beat to beat variation and orthostatic hypotension test) are fully validated and acceptable according to present consensus, thus fulfilling criterion e), this is not the case for peripheral nerve function. Measurement of vibration threshold with a biothesiometer as described is a simple bedside test for evaluating peripheral nerve function with a high retest reliability making it suitable for follow-up studies (86). However, it only studies the unmyelinised C-fibre qualities, whereas the fastest and more important myelinised A-fibres are not assessed. Since sensory examination in criterion d) requires both examination of hypaesthesia, hypalgesia, and vibration threshold, this criterion is only partly fulfilled. Criterion a), b), and c) have not been met. Our finding of a lack of effect of intensified multifactorial intervention on peripheral nerve function in microalbuminuric patients with type 2 diabetes both after four and eight years of follow-up should be seen in the light of this weakness in our methods for measurement of peripheral neuropathy (44; 46). Also, it should be underlined that development or progression of diabetic neuropathy was a secondary and tertiary endpoint, respectively.

4.7 Cardiovascular disease

Past and present symptoms of cardiovascular disease were registered according to the World Health Organization cardiovascular questionnaire (87).

A 12-lead resting ECG was recorded. The ECG was coded independently by two trained, masked observers using the Minnesota Rating Scale (88). Minnesota codes 1.1-1.3, 4.1-4.4, 5.1-5.8 and 7.1 were taken as sign of cardiac ischaemia.

Exercise stress test was done with a bicycle ergometer (Kivex, Copenhagen, Denmark) beginning at 25 W and increasing the work load with 25 W every second minute. All electrocardiograms were evaluated by two independent, masked graders. Ischaemia was present if ST-depression was greater than 1 mm in any lead.

Digital systolic blood pressure in the lower limb was measured in the first toe using a strain gauge technique (89).

An independent, masked endpoint committee consisting of 2 specialists in cardiology and one specialist in diabetology evaluated all cases and classified cardiovascular events into the following categories: cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, amputations, invasive cardiovascular procedures, and peripheral vascular procedures (Appendix 1).

The role of plasma NT-proBNP as a risk factor for cardiovascular disease and heart failure was also studied in the Steno-2 cohort (49). A secondary endpoint comprising cardiovascular mortality as defined above as well as heart failure was examined. Heart failure was defined as admission for heart failure documented by discharge letters.

5.1 Lifestyle education

The underlying theories and the practical approach to lifestyle education about diet, exercise and smoking in the Steno-2 study are discussed in detail in one of the reports from the study (45).

5.2 Drug therapy

The following is a summary of the drug treatment strategy in the intensive therapy group in the Steno-2 study (44; 46). Treatment was target driven according to the overall treatment goals set in the study protocol, yet in order to increase adherence to the various drug treatments, we chose to use a stepwise implementation based on individual risk assessment and individualised intermediate goals.

5.2.1 Hyperglycaemia

The goal for blood glucose was a glycosylated haemoglobin A 1c (HbA 1c ) below 6.5%. If patients were unable to maintain HbA 1c values below 6.5 percent on diet and increased physical activity alone after 3 months, treatment with oral hypoglycaemic agents was started. As the initial step overweight patients (body mass index (BMI) > 25 kg/m 2 ) received metformin to a maximum of 1 g twice daily; lean patients, or overweight patients with contraindications to metformin, received gliclazide to a maximum of 160 mg twice daily. As the second step metformin was added to lean and gliclazide to overweight patients if hyperglycaemia was not controlled. If HbA 1c was above 7.0 percent despite maximum doses of oral agents the addition of neutral protamine Hagedorn (NPH) insulin at bedtime was recommended. When insulin was started lean patients stopped metformin treatment and overweight patients stopped gliclazide unless this was the only oral hypoglycaemic agent given. The insulin dose was adjusted by the morning fasting blood glucose concentration (90). If the average fasting blood glucose exceeded 7 mmol/l during a three day period, patients increased the evening NPH insulin dose with four units until the fasting blood glucose had reached target. If the daily insulin dose exceeded 80 IU or there was no decrease in HbA 1c patients were switched to insulin regimens with NPH insulin given two times a day or short acting insulin to main meals and NPH insulin at bedtime. There was no upper limit for the daily insulin dose.

5.2.2 Hypertension

During the first six years of the study the goal for blood pressure was 140/85 mm Hg, but since newer and stricter guidelines were applied to the conventional group during the last two years of the study, the goal in the intensive therapy group was intensified to 130/80 mm Hg during this period. Our first line strategy was blockade of the renin-angiotensin system with the angiotensin converting enzyme inhibitor captopril 50 mg twice daily. In case of side effects the angiotensin II receptor antagonist losartan 50 mg twice daily was given. During the last two years of the study we had the possibility of combining the two drugs (91). If goals were not met the second step was addition of diuretics. Depending on whether patients had oedema or not furosemide with an initial dose of 40 mg daily or bendroflumethiazide 5 mg administered once daily was prescribed. The third step was addition of the long-acting dihydropyridine calcium antagonist amlodipine 10 mg given once daily. The beta-blocker metoprolol with a maximum dose of 200 mg per day was the fourth step in treating uncontrolled hypertension in the intensive therapy group.

5.2.3 Dyslipidaemia

The goal for treatment of dyslipidaemia was based on fasting serum levels of total cholesterol and triglycerides. The goal for fasting serum total cholesterol was 5.0 mmol/l during the first six years of the study with a tighter goal of 4.5 mmol/l during the last two years. The goal for fasting serum triglycerides was 1.7 mmol/l throughout the entire study period. Initially fluvastatin was used, but since atorvastatin became available in Denmark during the last four years of the study this drug was used. The dose was titrated based on fasting serum total cholesterol levels to a maximum of atorvastatin 40 mg per day. In case of an elevated fasting serum triglyceride level above 4.0 mmol/l despite treatment with a statin, the fibrate gemfibrozil in a maximal dose of 600 mg twice daily was used in combination with the statin.

5.2.4 Microalbuminuria

All 160 patients included in the Steno-2 study had microalbuminuria. Based on the early findings from the study by Ravid et al (92), where beneficial effects on kidney function were seen in normotensive patients with type 2 diabetes, all patients in the intensive therapy arm were given ACE inhibition with captopril 50 mg twice daily irrespective of blood pressure level. In case of side effects the angiotensin II receptor antagonist losartan 50 mg twice daily was prescribed.

5.2.5 Acetylsalicylic acid

The use of low-dose acetylsalicylic acid (ASA) was quite extensive throughout the entire study period. During the first six years ASA 150 mg daily was given as secondary prevention to patients with a history of a) transitory ischaemic attack, b) stroke, c) myocardial infarction, d) signs of ischaemic heart disease, and e) systolic toe/brachial blood pressure index below 0.67. During the last two years of the study ASA 150 mg daily was recommended as primary prevention to all patients in the intensive therapy group unless contraindications were present.

5.2.6 Vitamins

As discussed in a later chapter the use of vitamins and minerals in the prevention and treatment of late diabetic complications is controversial. During the first four years of the study patients were recommended a tablet consisting of vitamin C 250 mg and vitamin E (d- α -tochopherol) 100 mg. The recommended dose was one tablet daily for non-smokers and five daily tablets for smokers. Furthermore, one multivitamin tablet was recommended to all patients in the intensive therapy group. During the last four years of the study the daily recommendations also consisted of chromium piccolinate 100 μ g and folic acid 400 μ g daily.

6.1 Risk factors for cardiovascular disease in type 2 diabetes

Recently, a major case-control study (the INTERHEART study) investigated the effect of potentially modifiable risk factors associated with myocardial infarction in almost 30,000 subjects from 52 countries (93). The conclusion was that current smoking, lipid abnormalities, hypertension, abdominal obesity, psychosocial factors, and diabetes were associated with an increased risk of myocardial infarction, while daily consumption of fruits and vegetables, regular alcohol consumption, and regular physical activity decreased the risk. These nine modifiable risk factors accounted for more than 90% of the risk for a myocardial infarction in the population. A similar large-scale study has not been performed in patients with type 2 diabetes, but several risk factors have been identified.

6.1.1 The classic risk factors

Data from prospective observational studies indicate that the major cardiovascular risk factors in the non-diabetic population, that is smoking, hypertension and dyslipidaemia, also operate in diabetic subjects (2; 94). Also genetic factors are important since the prevalence of cardiovascular disease (CVD) is influenced by the population itself (95). In the Multiple Risk Factor Intervention Trial (MRFIT) (2) more than 5,000 men with type 2 diabetes and 340,000 non-diabetic men were followed for 12 years. The risk for cardiovascular mortality increased significantly with the number of risk factors (systolic blood pressure, total serum cholesterol, and smoking) in both the diabetic and non-diabetic men, but the risk of cardiovascular death was two to three-fold increased in the diabetic population for each combination of risk factors. Increased systolic blood pressure is more common in type 2 diabetes than in the general population (3; 96). In the UKPDS observational study a 10 mm Hg decrease in updated mean systolic blood pressure during treatment was associated with a significant 11% relative reduction in the risk for myocardial infarction, a 19% relative reduction in the risk for stroke, and a 15% relative reduction in the risk for diabetes related death (97). It should, however be noted, that the strongest risk factor for CVD in the UKPDS was dyslipidaemia with estimated hazard ratios for the upper third relative to the lower third of 2.26 for fasting serum LDL-cholesterol, and 0.55 for HDL-cholesterol (94). In the same study, current smoking was of borderline significance only. Since the classic risk factors do not explain all of the excess cardiovascular mortality in patients with diabetes other risk factors must be of importance (98; 99).

6.1.2 Hyperglycaemia

Several prospective studies have shown that in type 2 diabetes hyperglycaemia increases the risk for myocardial infarction (94; 100; 101), stroke (102), macrovascular mortality (103-105), and all-cause mortality (103; 106-109). It should here be mentioned that many earlier studies have been hampered by the lack of valid estimates of long-term glycaemic regulation. Measurement of glycosylated haemoglobin concentrations yield such estimates, however the method has only been available for the last 20 years and as a consequence, many previous studies have been based on measurements of fasting or random plasma glucose levels, which are less accurate in estimating long-term glycaemic regulation.

6.1.3 Hyperinsulinaemia

Hyperinsulinaemia has generally been considered a marker of insulin resistance, i.e. a decrease in the effect of insulin to stimulate glucose uptake at a given serum insulin concentration. Since high serum insulin concentrations in animal studies stimulate cholesterol synthesis and binding of LDL-cholesterol to smooth muscle cells and macrophages in the arterial wall (110) a causative link between elevated levels of serum insulin and the risk for cardiovascular disease may exist. Such an association has been found both in non-diabetic men (111; 112) and in type 2 diabetic patients (113). In 1988 Reaven introduced the term Syndrome X or the metabolic syndrome and suggested that insulin resistance and compensatory hyperinsulinaemia may underlie the clustering of cardiovascular risk factors seen in type 2 diabetes as a possible mechanism for the increased risk for CVD in these patients (114). In a recent study from Finland the clustering of a high BMI, high fasting serum triglyceride concentration, low fasting serum HDL cholesterol and hyperinsulinaemia predicted cardiovascular mortality in type 2 diabetic patients who were not treated with insulin (115).

6.1.4 Dyslipidaemia

It has long been known that more than half of patients with newly diagnosed type 2 diabetes have dyslipidaemia (116). The typical pattern is elevated levels of fasting serum triglycerides, decreased levels of fasting serum HDL-cholesterol, a predominance of small dense LDL particles, and exaggerated postprandial lipidaemia (117). Epidemiological studies have established a direct proportional relation between the fasting serum concentrations of total-cholesterol or triglyceride and the risk for ischaemic heart disease in type 2 diabetes (2). The importance of dyslipidaemia in predicting cardiovascular disease in patients with type 2 diabetes is also seen from the UKPDS, where the most important predictor for CVD during a nine year period was increased fasting serum LDL-cholesterol followed by decreased fasting serum HDL-cholesterol concentration, as mentioned previously (94).

6.1.5 Microalbuminuria

In 1984 both Mogensen (118) and Jarret (119) reported independently that microalbuminuria predicted all-cause mortality in type 2 diabetes. These findings have later been found also to extend to CVD morbidity in both men and women (120). Thus, an extensive review of the literature carried out by Dinneen and Gerstein (38) has confirmed the strong association between microalbuminuria and cardiovascular mortality in type 2 diabetes. Interestingly, this association between high levels of UAER and cardiovascular disease and mortality has been shown also to extend to the non-diabetic population, even within the normal range of UAER as is also the case for patients with type 2 diabetes (121-124). Why the development of microalbuminuria, in itself reflecting a trivial loss of albumin, should herald such serious and anatomically far reaching consequences is not understood. In an attempt to explain this, Deckert et al have put forward the hypothesis that increased UAER loss merely reflects a glomerular manifestation of an otherwise generalised (but less clinically visible) vascular hyperpermeability state (125). Additional plausible explanations might be the association between microalbuminuria and insulin resistance and the components of the insulin resistance syndrome in type 2 diabetes (126; 127).

6.1.6 Hypercoagulation and endothelial dysfunction

An imbalance in the haemostatic system due to hypercoagulability or impaired fibrinolytic function may favour the development of vascular damage. Plasminogen activator inhibitor type 1 (PAI-1) is a potent inhibitor of fibrinolysis, and increased plasma levels of PAI-1 have been demonstrated in patients with coronary artery stenosis or after acute myocardial infarction (128). Epidemiological studies have suggested links between plasma PAI-1 levels and the components of the metabolic syndrome (129). Fibrinogen is another player in the coagulation system and raised circulating concentrations favour coagulation, increase platelet activation and adherence to the endothelium, and have been associated with CVD in the general population (130). In type 2 diabetes a similar association has been shown with circulating fibrinogen levels increasing with age, hyperglycaemia, smoking, hypertension and other components of the metabolic syndrome (131). Elevated levels of plasma von Willebrand factor have also been associated with increased cardiovascular mortality in type 2 diabetes (104) and some prospective studies even suggest that the role of microalbuminuria in predicting CVD in type 2 diabetic patients is largely influenced by the absence or presence of endothelial dysfunction as measured by elevated plasma levels of vWF (132; 133). We also examined this concept in a post-hoc follow-up study lasting for an average of 3.8 years in the 160 microalbuminuric patients participating in the Steno-2 study (48). Patients were divided into two groups according to plasma vWF levels below or above the median at baseline. Although the odds ratio for cardiovascular disease was 1.11 with elevated plasma vWF this difference was not significant in our setting.

6.1.7 N-terminal-proBrain Natriuretic Peptide

Brain natriuretic peptide (BNP) is synthesised by cardiocytes as a response to increased cardiac wall stress and mediates natriuresis, diuresis and vasodilatation (134). BNP is synthesized as a prohormone which is cleaved into BNP and N-terminal proBNP (NT-proBNP), the latter being more stable in vitro with a longer half-life. The role of BNP as a prognostic risk marker for CVD has been investigated in patients with chronic heart failure and acute myocardial infarction showing increased risk for future CVD morbidity or mortality with elevated plasma levels of BNP. Measurement of plasma NT-proBNP seems to provide the same information as plasma BNP (135). The role of plasma NT-proBNP as a risk marker for CVD was examined in the Steno-2 cohort (49). In this study sample the range of fasting plasma levels of NT-proBNP at baseline was 5.0 (lowest detectable value) to 1290.0 pg/ml with a median value of 33.5 pg/ml. Interestingly, the level of plasma NT-proBNP was low in the type 2 diabetic patients with microalbuminuria included in the Steno-2 Study, thereby expanding the use of NT-proBNP as a risk marker for future CVD to levels seen in the general population (136). Plasma NT-proBNP levels above the median were significantly correlated with an increased risk of CVD as defined in the Steno-2 study (Hazard ratio 4.4 (95% confidence interval 2.3-8.4), (p < 0.0001)) as well as a secondary combined endpoint of cardiovascular mortality and hospitalization for heart failure (Hazard ratio 5.8 (2.0-16.9), p=0.001). The association between elevated levels of plasma NT-proBNP and prognostic outcomes was also seen when each of the two original treatment groups (intensive therapy or conventional therapy) was analysed separately (49).

6.1.8 Other risk factors

Although lack of physical activity predicts CVD in non-diabetic individuals (93; 137), data in diabetic patients are limited. Yet, a low level of physical activity has been associated with increased risk for CVD in men with type 2 diabetes (138). Obesity, a very common characteristic of type 2 diabetes, has not independently been associated with CVD in diabetic patients (139; 140). Still, central obesity predicts CVD in prospective studies independently of overall obesity in men with type 2 diabetes (141).

6.2 Risk factors for microvascular complications in type 2 diabetes

Several studies have investigated the relationship between putative risk factors and diabetic nephropathy, retinopathy, and neuropathy (17; 19; 42; 142-167). Table 1 gives a brief summary of current knowledge in this area. As seen from this table with selected risk factors many of the risk factors known to have impact on the development of CVD also play an important role in the development of microvascular complications. The associations between hyperglycaemia and both diabetic nephropathy, retinopathy and neuropathy seem quite consistent while evidence is far less convincing for some of the other risk factors.

7.1 Lifestyle interventions

7.1.1 Diet intervention

The rationale for diet intervention in type 2 diabetes is obvious. Since dietary intervention in short term trials has been shown to reduce several risk factors for both macro- and microvascular complications it remains a cornerstone in the treatment. The effects of diet intervention is either direct from diet itself or indirect from the effect on weight and body composition. It must, however, be emphasised that the benefits of this kind of intervention in reducing complications has never been proven in randomised long-term studies in type 2 diabetes. The following paragraphs will discuss the effect of changing diet upon different risk factors.

7.1.2 Exercise

The possible benefits of exercise for the patient with type 2 diabetes are substantial since in epidemiological studies positive effects of exercise are seen on several risk factors such as hyperglycaemia, dyslipidaemia, and hypertension. As for diet the effects are mediated either by exercise itself or by changes in weight and body composition. Yet, no randomised studies have documented any effects on macro- or microvascular complications in these patients.

7.2 Overweight

Since overweight and obesity are strong predictors for the development of type 2 diabetes it seems obvious that treatment of overweight will have beneficial effects in type 2 diabetic patients. Weight loss has in epidemiological studies been associated with a reduction in insulin resistance and an improvement in risk factors for macro- and microvascular disease in type 2 diabetes (187). The size of the weight reduction in order to achieve clinically relevant changes in risk factors is, however, quite large around 15% of body weight. Furthermore, in intervention studies with behavioural weight-control programs it seems that type 2 diabetic patients loose less weight than their overweight non-diabetic spouses (188). Another problem in inducing weight loss in type 2 diabetic patients is the lack of studies demonstrating that the reductions in weight seen during short-term programs can be maintained in the long-term (189). A meta-analysis including 89 studies and 1800 patients with type 2 diabetes comprising studies with a duration of up to one year has investigated the effect of different treatment strategies in reducing weight in this type of patients (190). All interventions except anorectic drugs given without behaviour therapies led to reductions in mean body weight. Dietary strategies led to a mean decrease in body weight of 9 kg and were associated with the largest changes in HbA 1c (2.7%). Surgery had the greatest effect on weight loss with an average weight loss of 22 kg, however this result was not obtained in a randomised study. Similarly, an average weight loss of 28 kg over a ten year period with gastric surgery in overweight patients with an average BMI of 41 kg/m 2 was seen in a Swedish study with beneficial effects on hyperglycaemia and hypertension compared to a matched overweight control group receiving conventional obesity treatment with dietary advice and anorectic drugs (191; 192).

A typical finding in randomised intervention studies examining the effect of intensive blood glucose lowering with oral hypoglycaemic agents or insulin is a weight increase following treatment with these drugs (44; 193-195). Although an increase in weight in type 2 diabetic patients is associated with deleterious effects on insulin sensitivity and aggravation of other risk factors in type 2 diabetes it should be emphasised that blood glucose lowering treatment alone (193; 195), or in combination with other treatments (44; 46) reduces the risk of long-term complications and as a consequence such a treatment should not be postponed or stopped because of fear of weight gain. Furthermore, we have shown that using a continuous behaviour modification strategy over an eight year period, the weight gain with intensive therapy was not significantly larger than with conventional therapy (46).

In summary, a substantial weight loss is needed to normalise risk factors in type 2 diabetes. Although induced weight changes are rarely of long lasting duration intervention may reduce the weight gain otherwise seen with intensified intervention against hyperglycaemia in type 2 diabetes. Extremely obese patients may benefit from gastric surgery.

7.3 Smoking cessation

As mentioned previously several studies have demonstrated a close association between smoking and risk for CVD in both the diabetic and the non-diabetic population (196). In that respect, it is disappointing that at this time there are no randomised, controlled intervention studies that have documented the beneficial effect of giving up smoking for patients with type 2 diabetes. The most comprehensive and successful intervention study performed to date, which included both non-diabetic and diabetic patients, was the MRFIT in which 12,866 men with a high risk of developing CVD were randomised to specific intervention against multiple risk factors (smoking, hypertension, hypercholesterolemia) at a medical centre or follow-up by the general practitioner (GP) with standard intervention according to generally accepted guidelines (197). After an average follow-up period of 7 years, 50% of the men in the intervention group had stopped smoking, while the equivalent percentage in the control group was 29. Already after one year, the two groups showed significant differences in the number of smokers in the groups. Despite this large difference in the number of smokers, no significant difference in the number of deaths caused by either CVD or cancer was found. No analyses of subgroups for diabetic patients have been published. There are no obvious explanations for this disappointing result. However, one explanation could be that the study did not have sufficient power to detect a difference in the follow-up time given. Another and quite interesting aspect is that the effect of smoking cessation on CVD may be lesser the longer the duration of smoking prior to cessation. This has in epidemiological studies been demonstrated to be the case in a mixed diabetes population and most recently in women with type 2 diabetes from the Nurses' Health Study cohort (198; 199). In the mixed diabetes population study 4,427 patients were followed. All-cause mortality risks were significantly higher for recent quitters (within 1 to 9 years) with a relative risk of 1.53 compared with patients who had never smoked. In comparison, those who had quit earlier (≥ 10 years) had a relative risk of 1.25 compared to patients who had never smoked. Also, the mortality rate was highest in those who had smoked the longest. In the latter study 7,401 women with type 2 diabetes were followed for 20 years (199). A clear dose dependent relationship between smoking and mortality risk was seen. The overall relative risk compared to never smokers was 1.31 for past smokers compared to never smokers, 1.43 for current smokers of 1-14 cigarettes per day, 1.64 for current smokers of 15-34 cigarettes per day, and 2.19 for current smokers of more than 34 cigarettes per day. Also in this study it was found, that patients who had stopped smoking more than ten years ago still had a significantly higher risk for mortality (relative risk 1.11) than patients who had never smoked. These two studies clearly indicate, that individuals with diabetes who smoke should be encouraged to quit as soon as possible in the course of the disease.

The majority of scientific papers about diabetes and smoking has focused on reviews of the current literature and have extrapolated from other studies to include issues of particular pertinence to diabetes (196). No randomised intervention studies in type 2 diabetes have addressed the efficacy of various smoking cessation strategies. In type 1 diabetes two randomised studies found equal effect of simple advice given by a physician as compared to more sophisticated behaviour intervention strategies not using nicotine replacement therapy (200; 201). In a meta-analysis of 53 randomised trials using nicotine replacement therapy with a follow-up of at least six months this approach doubled the chance of smoking cessation, but none of the studies included reported effects from type 2 diabetic patients (202).

In the Steno-2 study we used a combination of behaviour modification strategies as well as nicotine replacement therapy in our smoking cessation programs for patients randomised to intensive multifactorial intervention. When evaluated two years after the last of two smoking cessation courses held during the trial, the smoking cessation rate in patients participating in these courses was 43% (45). In comparison, this rate has been found to be approximately 18% one year after smoking cessation in several other studies (203). However, the number of smokers was not significantly reduced in the intensive as compared to the conventional group at four or eight years after study start (44; 46).

In conclusion, although the definite proof for and size of the beneficial effects from smoking cessation need to be investigated in randomised trials, overwhelming epidemiological evidence suggests that all patients with type 2 diabetes should refrain from smoking. Since the deleterious effects of smoking persists more than ten years after quitting smoking, smoking cessation should be encouraged early in the course of the disease. However, even in patients who smoke, late diabetic complications can be reduced with intensified multifactorial intervention (46).

7.4 Pharmacological interventions

An extensive review of the many single risk factor intervention studies with special emphasis on patients with type 2 diabetes has recently been published (204). Tables 2-6 summarise the major randomised intervention studies with single risk factor treatment of hyperglycaemia, hypertension, dyslipidaemia and microalbuminuria. Preventive treatment with low-dose acetylsalicylic acid, ACE inhibitors, and treatment with vitamin C and E will be discussed below as well as certain features from single risk factor intervention trials with special relation to the interventions given in the Steno-2 study.

7.4.1 Pharmacological treatment of hyperglycaemia

Single risk factor trials intervening against hyperglycaemia are shown in Table 2. Although hyperglycaemia is a strong risk factor for micro- and macroangiopathy in type 2 diabetes, intervention against hyperglycaemia in randomised trials has only demonstrated clear effects of intervention on microangiopathy ( Table 2 ). It is, however, of importance to notice that the blood glucose levels obtained were higher than targets according to national guidelines. However, metformin seems to pose special benefits in overweight or obese diabetic patients, yet a possible deleterious effect of this drug in patients with secondary failure to sulphonylureas needs to be elucidated.

7.4.2 Pharmacological treatment of hypertension

Besides the obvious question whether treatment of elevated blood pressure in type 2 diabetic patients reduces the risk of complications, two questions with clinical relevance concerning antihypertensive treatment of patients with type 2 diabetes should be addressed: 1) what is the desired blood pressure? (53; 205-207) and 2) which antihypertensive drug should be prescribed? (54; 208-214). Table 3 summarises some of these studies, clearly demonstrating the benefits of lowering blood pressure in patients with type 2 diabetes.

7.4.3 Pharmacological treatment of dyslipidaemia

The use of pharmacological intervention with statins in the prevention of CVD has been extensively examined, especially in patients with previous known ischaemic heart disease (secondary prevention) (217-226). However, only one study has investigated the effect of primary pharmacological intervention with statins solely in patients with type 2 diabetes (227), and only a few have investigated the effects of fibrates as secondary intervention (228-230), and as a consequence current recommendations are based on subanalyses from larger studies including patients with diabetes. Results from these studies are summarised in Table 4 .

The above mentioned trials have with the exception of one (224) prescribed a fixed statin dose, thus leaving open the question of the optimal dose for the different statins. Furthermore, studies comparing different statins in equipotent doses are also missing. One study has compared the effect of intensive versus moderate lipid lowering treatment after acute myocardial infarction using two different statins in non-equipotent doses. The Pravastatin or Atorvastatin Evaluation and Infection Therapy - Thrombolysis in Myocardial Infarction 22 (PROVE IT - TIMI 22) trial compared the effect of 40 mg of pravastatin daily with 80 mg of atorvastatin daily in 4,162 patients with an acute coronary syndrome (231). The primary endpoint consisted of all cause mortality, myocardial infarction, documented unstable angina requiring rehospitalisation, revascularisation, and stroke. Eighteen percent of patients included had diabetes at randomisation. During a follow-up of two years 26.3% of patients in the standard dose pravastatin group had an event compared to 22.4% in the high-dose atorvastatin group, representing a 16% relative risk reduction in the hazard ratio favouring atorvastatin, p=0.005. The difference was seen already after 30 days of intervention. Although the risk reduction with high-dose atorvastatin was consistent among several previous specified subgroups, it was not significant in patients with diabetes (HR 0.81 (95% confidence interval 0.62-1.03)). The difference in fasting serum LDL-cholesterol was about 1 mmol/l throughout the study period.

To conclude, post hoc subgroup analyses of patients with type 2 diabetes mellitus and known ischaemic heart disease with normal or raised fasting serum total cholesterol values or too low fasting serum HDL-cholesterol values have documented the beneficial effect of secondary prevention using statins or fibrates. The effect of fibrates as primary prevention of ischaemic vascular disease has not been documented. A significant effect of simvastatin as primary prevention in the subgroup of patients with diabetes and a non-fasting serum cholesterol level above 3.5 mmol/l has been shown in the Heart Protection Study (232), while the Collaborative Atorvastatin Diabetes Study found a significant effect of primary prevention with atorvastatin in type 2 diabetic patients with modest elevations of fasting serum LDL-cholesterol (227). As a consequence these findings have triggered the discussion whether all patients with diabetes should be given statin treatment as primary prevention. If it can be replicated, that the absolute risk reductions for coronary heart disease with statin treatment is the same throughout the spectre of fasting serum cholesterol concentrations as suggested in the Heart Protection Study (HPS) (232) as well as in the Collaborative Atorvastatin Diabetes Study (CARDS) (227), it indeed seems favourable to give all patients with type 2 diabetes a statin as primary prevention. However, it should be mentioned that the subgroup analyses in patients with diabetes in both the Anglo-Scandinavian Cardiac Outcomes Trial-Lipid Lowering Arm (ASCOT-LLA) (225) and the The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT-LLT) (226) did not show significant risk reductions with statin treatment as primary prevention. However, the current evidence based on less rigorous inclusion criteria in the studies suggests that patients with type 2 diabetes should be treated with a statin as primary prevention unless their risk for cardiovascular disease is found sufficiently low to withhold such a treatment ( Figure 2 ).

Fasting serum LDL-cholesterol levels serve in most guidelines as an indicator for cholesterol-lowering therapy, since a linear relationship between reduction in fasting serum LDL-cholesterol level and the size of risk reduction in CVD has been demonstrated (233). However, large clinical trials indicates that statin-treated individuals have significantly less CVD than patients with comparable serum cholesterol levels (221). Experimental data have shown that statins exhibit pleiotropic effects that can beneficially impact occlusive vascular disease, including inhibition of smooth muscle proliferation and platelet aggregation, enhancement of endothelial function, and antiinflammatory actions (234-236). Thus, there appears to be a growing list of actions that are attributed to statins beyond their ability to reduce serum cholesterol levels. It remains to be determined, however, which, if any, of these effects are actually clinically important at the dose range used.

In the discussion whether treatment should be simvastatin or another statin it should be recalled, that the effect of simvastatin has been proven in a single risk factor intervention trial. The typical type 2 diabetic patient will take several drugs to diminish the impact of several risk factors. In the Steno-2 study where multiple risk factor intervention in type 2 diabetic patients with and without known CVD at baseline was associated with an absolute risk reduction of 20% for CVD during eight years, the statin used was atorvastatin (46).

7.4.4 Specific treatment of elevated urinary albumin excretion rate in type 2 diabetes

Specific treatment of microalbuminuria (UAER between 30 and 300 mg per 24 hour) with ACE inhibitors in intervention studies of patients with type 1 diabetes have proven to possess a albuminuria reducing effect in normotensive as well as hypertensive patients, an effect found to be independent of the antihypertensive effect of the ACE inhibitors (237; 238).

Even though a significant risk reduction in developing dialysis-dependent kidney disease or cardiovascular morbidity and mortality following treatment with ACE inhibitors has not yet been established, ACE inhibitors are now widely used as standard intervention in patients with type 1 as well as type 2 diabetes complicated by microalbuminuria, regardless of the presence of hypertension. Studies that form the evidence for the treatment effect of blocking the renin-angiotensin system are summarised in Table 5 (92; 211; 239-241).

To conclude, there is strong evidence that the angiotensin II receptor antagonists pose albuminuria reducing effects beyond that of blood pressure lowering alone in type 2 diabetes. Whether a similar effect of ACE inhibitors exists is not known. No studies have compared ACE inhibitors and angiotensin II receptor antagonist head-to-head in a randomised trial in type 2 diabetes. In our Steno-2 study all patients in the intensive therapy group were prescribed an ACE inhibitor (captopril) because of the presence of microalbuminuria. Significant reductions in the risk for nephropathy were seen both after four and eight years of intervention, respectively, with intensive compared to conventional treatment. However, while none of the patients received combined treatment with an ACE inhibitor and an angiotensin II receptor antagonist after four years, this was the case for 28% of the patients in the intensive arm and for none of the patients in the conventional treatment arm at the end of the trial.

7.4.5 Preventive treatment using low-dose acetylsalicylic acid

The value of ASA as secondary cardiovascular prevention in diabetes is indisputable (242). As seen from Table 6 primary prevention treatment with low-dose ASA in diabetic patients showed an effect in three of the reported studies described (243-247). By inhibiting platelet activation treatment with low-dose ASA is associated with a significantly increased risk of gastrointestinal bleedings requiring transfusions as well as epistaxis, haematuria, and easy bruising. Absolute risk reductions are larger in high risk patients, thereby diminishing the role of side-effects. As a consequence, low-dose ASA should only be considered as primary prevention to patients with type 2 diabetes having a Framingham point score (appendix 2) above 0.6% per year (248).

7.4.6 Preventive treatment with ACE inhibitors

The Heart Outcomes Prevention Evaluation (HOPE) Study was based on the hypothesis that the renin-angiotensin-aldosterone system plays a vital role in the development of CVD (249). A total of 9,297 patients were randomised to double-blind treatment with ramipril or placebo. All patients included suffered from known CVD or diabetes with at least one additional risk factor (hypertension, increased fasting serum total-cholesterol, low fasting serum HDL-cholesterol, microalbuminuria or smoking) and were therefore at high risk of cardiovascular events. The primary endpoint consisted of a combination of myocardial infarction, stroke or death caused by cardiovascular disease. Also a 26% relative risk reduction (p=0.03) was found in the number of patients with diabetes-related complications, defined as 24-hour UAER > 300 mg or 24-hour urinary protein excretion > 500 mg, dialysis-dependent kidney insufficiency and photocoagulation caused by retinopathy. No results are available for this endpoint in terms of diabetic patients without diagnosed heart disease. A subgroup analysis in a separate paper reported a significant 25% relative risk reduction for the primary CVD endpoint with ramipril treatment compared with placebo in patients with diabetes (250). Since blood pressure did not differ significantly between treatment groups this difference cannot directly be explained by a difference of standard blood pressure measures in the main study. However, a subgroup analysis in 38 patients showed a significantly lower 24 hour blood pressure profile (10/4 mm Hg, p=0.03) in patients treated with ramipril compared to placebo (251). Similarly, when focusing only on diabetic patients without previously known cardiovascular disease but with at least one of the earlier mentioned risk factors, no significant difference between ramipril treatment and placebo was found.

An eightfold lower dose of ramipril was used in the the Non-insulin-dependent diabetes, hypertension, microalbuminuria or proteinuria, cardiovascular events, and ramipril (DIABHYCAR) study enrolling almost 5,000 patients with type 2 diabetes and persistent microalbuminuria or proteinuria (252). The primary endpoint was the combined incidence of cardiovascular death, non-fatal myocardial infarction, stroke, heart failure leading to hospital admission, and ESRD. Despite small reductions in both systolic and diastolic blood pressure during a median follow-up time of four years, no risk reductions were seen for the combined endpoint or the components of the endpoint. However, low-dose ramipril favoured regression from microalbuminuria and proteinuria to lower levels of UAER.

To conclude, based on the HOPE study it is recommended that diabetic patients should be offered treatment with ACE inhibitors as secondary prevention of CVD, while there is no evidence to support the use of ACE inhibitors as primary prevention of CVD.

7.4.7 Treatment with antioxidant vitamins E and C

8.1 Which conclusions can be drawn from multifactorial intervention trials?

Which conclusions can be drawn from these studies in trying to answer some of the questions mentioned earlier? First, all six studies demonstrated that risk factor intervention against several concomitant risk factors can be done both at the level of general practitioners and at diabetes clinics with the Steno-2 study being the most successful in the number of risk factors reduced. Secondly, both the Steno-2 study and the study from Israel demonstrated benefits on morbidity of intensified multifactorial intervention with significant risk reductions of both cardiovascular disease as well as microvascular complications. This was, however, not the case for the two studies including patients with newly diagnosed type 2 diabetes. An obvious explanation for this discrepancy is the much higher number of risk factors reduced in the Steno-2 study, and the much larger reductions in these risk factors with intensified therapy in this study. Other studies used guidelines with risk factor targets for the intervention groups that were similar to the ones used in the control groups, thereby in itself diminishing the effect of intensified intervention. Also, a time gap from new pieces of clinical evidence are made until implementation in national guidelines will of course always exist, withholding patients in the intervention groups the benefits of new knowledge. As an example the optimal goal for fasting serum total cholesterol in the structured care group in Diabetes Care in General Practice was 6 mmol/l although several studies indicated that a much lower value should be strived for (25). Similarly, the goal for systolic and diastolic blood pressure was set high in this as well as in the Diabetes Intervention Study (170). As discussed in an earlier chapter intervention against dyslipidaemia and blood pressure has proven to have a pronounced impact as single risk factor interventions in patients with type 2 diabetes, whereas intervention against hyperglycaemia was not as effective as expected from epidemiological studies. As a consequence, one explanation for the lack of effect of intensified multifactorial intervention in patients with newly diagnosed type 2 diabetes is that these studies have not been sufficiently aggressive and proactive in their target settings.

Patients in the Steno-2 study were at higher risk for late diabetic complications than patients in the other studies, since patients in the Steno-2 study were selected because of microalbuminuria, thereby increasing the statistical power to detect differences in endpoints between the two treatment groups. Thus, with our present knowledge the questions a) and b) asked in the beginning of the chapter cannot be fully answered. Clear benefits of multifactorial intervention have been shown in a subgroup of patients with elevated UAER. These patients may comprise up to one third of patients with type 2 diabetes (16). However, further studies are needed before intensified multifactorial intervention can be recommended to patients with newly diagnosed type 2 diabetes, unless of course other important risk factors are present. It is of outmost importance, that the study design of new studies will allow sufficient separation in many risk factors between treatment groups.

The question whether an intensified risk factor intervention can be given at the level of general practitioners has also been confirmatory answered (25; 258; 260). However, a recent cross sectional study from the UK has demonstrated that it is absolutely essential to increase focus and intensify treatment of type 2 diabetes in general practice (261). A population of almost 8,000 patients with type 2 diabetes attending a total number of 42 general practices were included in the study. Thirty-one percent of all patients with type 2 diabetes were treated with diet only. More than four-fold variation between practices existed (range 15.6-73.2%). Patients treated with diet only were much less likely to have HbA 1c measurements, blood pressure, cholesterol, smoking, microalbuminuria testing, or screening for foot pulses recorded. Thirty-eight percent of patients with type 2 diabetes on medication had an HbA 1c above 7.5%. Compared with those on medication, patients treated by diet only were more likely to have raised blood pressure and less likely to be on antihypertensive medication. They were 45% more likely to have raised serum cholesterol levels and less likely to be prescribed lipid-lowering medication.

The success of a treatment strategy depends both on the patient's ability or will to adhere to the treatment prescribed as well as possible physician barriers against the treatment. Studies have shown that only 50 to 70% of the prescribed medication is actually taken by patients (262; 263). Furthermore, it has been clearly demonstrated that poor adherers are much more likely to have a bad outcome whether they are taking active study medication or placebo (264). Several factors are believed to be important for drug adherence. Many of the therapies given in an intensified multifactorial intervention approach are given as preventive treatments irrespective of the presence of symptoms, and therefore patients without symptoms may find, that the treatment may interfere more with daily life than the disease itself. In this respect, it is worth noticing, that patients may find that a change in lifestyle can lead to a large reduction in the quality of life and thus be a larger barrier for adherence to treatment than taking drugs (262). Even in case of symptoms, the start of a treatment may not relieve these, thereby in itself being a risk factor for non-adherence to treatment (265). However, another study did not find an association between relief of symptoms and adherence to treatment in a follow-up study for four years of more than 2,000 patients (266). The complexity of the drug regimen does also seem to be of importance, especially the number of dosages per day with decreased adherence the higher the number of dosages (267-269). Of course, side effects including drug interactions will also influence drug adherence, and finally cost of treatment may also be of importance. These issues will be discussed later. None of the three multiple risk factor intervention studies discussed had standardised measurements for adherence to treatment. The number and doses of drugs taken in each of the studies were by self-report by the patients.

In the Steno-2 study all patients in the intensive group were prescribed ACE inhibitors or angiotensin II receptor antagonists as well as a vitamin-mineral supplement which was delivered for free to the patients. While 97% of patients reported to take the ACE inhibitor or angiotensin II receptor antagonist at the eight year examination only 63% of patients reported to take the vitamin-mineral supplement. This shows that even within the same study group different barriers exist to different types of treatments. Clearly, the answer to question d) is that there indeed are major problems with adherence to both lifestyle and drug treatment in patients with type 2 diabetes. Further studies investigating solutions to these problems are warranted. Until these studies are available every effort must be made by health care personnel to make patients adherent to treatment according to current guidelines. One of the obstacles may be, that some physicians still think of type 2 diabetes as a relatively benign disease (270). It has been shown that physician barriers in following guidelines are related to the physicians knowledge of the disease (271). Fortunately, information to physicians about the disease can optimise the treatment of risk factors, as it has been demonstrated at the level of general practitioners (25; 258; 260).

Apart from being an obvious barrier to drug adherence side effects may cause serious health problems to patients. The magnitude of side effects of new drugs is investigated as part of the registration procedure, and furthermore most of the drugs mentioned in previous chapters have been investigated in single risk factor intervention trials. However, use of polypharmacy with several possible drug interactions has not been examined to a similar extend. One of the interactions that have been debated is the use of acetylsalicylic acid and ACE inhibitors, which is quite common in the treatment of type 2 diabetes. Some studies have suggested, that the beneficial effects of ACE inhibitors in reducing cardiovascular disease is diminished in patients taking acetylsalicylic acid (272), and also the combination of statin treatment and clopidogrel given as secondary prevention following a myocardial infarction and have been reported to weaken platelet inhibition (273). Even though these interactions may not play an important role at the clinical level, it does stress the importance of thorough investigation of side effects and drug interactions in patients treated with polypharmacy. Another example of concern is from the treatment of dyslipidaemia where beneficial treatment effects of both fibrates and statins have been demonstrated in single risk factor intervention trials. Yet, the combination of these two drug classes is not recommended, and recently one statin (cerivastatin) was withdrawn from the market because of fatal side effects when used in combination with a fibrate (274).

None of the six studies investigating the effect of intensified polypharmacological treatment in type 2 diabetes reported detailed information about drug interactions and side effects, and as a consequence our knowledge on this area is sparse. Further studies in this area are definitely required, although it is impossible to investigate all the possible drug combinations prescribed to patients with type 2 diabetes. This makes it, however, even more important with cautious follow-up of patients whenever new drugs are prescribed. One of the common side effects to treatment of hyperglycaemia in single risk factor trials is weight gain. It is noteworthy, that all six multiple risk factor intervention trials in type 2 diabetes found, that weight gain was, although expected, not significantly more pronounced with intensive than with conventional therapy. Similarly, hypoglycaemia was not more frequent with intensified multifactorial intervention compared to the control groups, although blood glucose levels were significantly lower with intensive therapy in all six studies.

The question of cost of treatment is also important, but results on this subject have not been published from the aforementioned multifactorial intervention studies in type 2 diabetes. The direct cost of drugs for the patient can definitely be a barrier for adherence to treatment (275). In Denmark reimbursement rules (year 2005) ensure that the direct cost of drugs cannot exceed DKK 3,805 per year (i 510). However, the cost of remedies and strips for blood glucose measurements, foot care, healthy food etc. are not included in this amount, since special reimbursement rules exist within this field. In the Steno-2 study all insulin treated patients in the intensive group were urged to measure blood glucose at least once daily in order to adjust insulin dose. Yet, patients' costs are only a fraction of the total cost. The total costs for an average Danish patient with type 2 diabetes have been estimated from Aarhus County based on a scenario, where most patients are followed at the level of GPs with an average of 3.6 consultations per year (276). These figures show that the typical patient is not treated according to current guidelines since for example the average numbers of eye examinations, consultations at a chiropodist, home blood glucose measurements, monitoring of UAER, and measurement of blood lipids are much lower than recommended. Similarly, one third of patients are not treated with glucose lowering drugs, and only 14% percent of patients are receiving cholesterol-lowering drugs. Despite this the total cost is estimated to i 750 with i 175 spent on health care personnel, i 460 spent on drugs, and i 115 spent on remedies and analyses. Although the cost-effectiveness of multifactorial intervention in patients with newly diagnosed type 2 diabetes has not been evaluated, the cost-effectiveness of single risk factor intervention against hyperglycaemia (277; 278), hypertension (279), and dyslipidaemia has been demonstrated in patients with type 2 diabetes (280; 281).

To summarise this issue, our current knowledge does not support an unrestricted use of intensified multifactorial intervention in type 2 diabetes if economic factors are considered a major issue. In contrast, the use of an intensified multifactorial approach may prove highly cost-effective in high risk populations, e.g. in type 2 diabetic patients with elevated albumin excretion rate, or with known cardiovascular disorders.

Appendix 1

Definitions of endpoints used in the Steno-2 study. An independent, masked endpoint committee consisting of two specialists in cardiology and one specialist in diabetology evaluated all cases and classified cardiovascular events into the following categories:

1.0 Cardiovascular death

1.1 Sudden death: Sudden death presumed to be due to ischaemic cardiovascular disease, occurring within 24 hours of the onset of symptoms without confirmation of cardiovascular disease, and without clinical or post mortem evidence of other aetiology.

1.2 Fatal myocardial infarction: death within 7 days of the onset of documented myocardial infarction (see 2.0).

1.3 Congestive heart failure: death due to clinical, radiological or post mortem evidence of congestive heart failure without clinical or post mortem evidence of an acute ischaemic event (which should then be coded as the cause). Cardiogenic shock to be included.

1.4 Post cardiovascular invasive interventions: death associated with the intervention: within 30 days of cardiovascular surgery, or within 7 days of cardiac catheterisation, or angioplasty, atherectomy, stent deployment or other invasive coronary or peripheral vascular interventions.

1.5 Documented arrhythmia: death due to bradyarrhythmias or tachyarrhythmias not induced by an acute ischaemic heart disease event (which should then be coded as the cause).

1.6 Death following non-cardiovascular surgery: death due to cardiovascular causes as defined in 1.1-1.5 and 1.7-1.8 and within 30 days of surgery.

1.7 Fatal stroke: death due to stroke occurring within 7 days of the signs and symptoms of a stroke.

1.8 Other cardiovascular diseases: death due to other vascular diseases including pulmonary emboli, and abdominal aortic aneurysm rupture.

1.9 Presumed cardiovascular death: suspicion of cardiovascular death with clinically supporting evidence which may not fulfil criteria otherwise stated. Example: Patient admitted with typical chest pain of 3 hours duration and treated as a myocardial infarction, but without ECG and enzymatic documentation to meet normal criteria.

2.0 Myocardial infarction (MI)

2.1 Q-wave MI: in comparison to the last ECG, presence of at least one new significant Q-wave on the standard 12-lead ECG as described in the Minnesota Code, and at least one of:

1. Typical symptoms (e.g. typical ischaemic chest pain lasting more than 30 minutes and/or

2. Significant elevation of serum enzymes - presence of any of the following criteria:

a) elevation of troponine to above the upper limit of normal for the laboratory that performed the test

b) elevation of creatine kinase MB (CK-MB) to twice the upper limit of normal for the laboratory that performed the test

c) elevation of total CK to at least twice the upper limit of normal for the laboratory that performed the test

d) elevation of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) to at least twice the upper limit of normal for the laboratory that performed the test with a characteristic pattern.

2.2 Non Q-wave MI: defined as a significant elevation of cardiac enzymes (at least twice the upper limit of normal) with or without characteristic pain in absence of new significant Q-wave.

2.3 Probable non Q-wave MI: presence of new and persistent ST-T changes (more than 24 hours in duration) on the ECG with characteristic symptoms of ischaemic chest pain without documentation of enzyme elevation.

2.4 Silent MI: development of new significant Q waves on the ECG (Minnesota Code) in at least two adjacent leads in the absence of any other evidence of myocardial infarction (in this case the date of event will be assessed as halfway between the date of discovery and last normal ECG).

2.5 Non-fatal MI post cardiovascular invasive interventions: MI (as defined in 2.1, 2.2, 2.3 or 2.4) associated with the intervention within 30 days of cardiovascular surgery, or within 7 days of cardiac catheterisation, or angioplasty, atherectomy, stent deployment or other invasive coronary or peripheral vascular interventions.

2.6 Non-fatal MI post non-cardiovascular surgery: MI (as defined in 2.1, 2.2, 2.3 or 2.4) occurring within 30 days of non-cardiovascular surgery.

3.0 Stroke

3.1 Definite ischaemic stroke: a CT or MRI scan within 2 weeks of onset of a definite stroke (focal neurological deficit greater than 24 hours) with evidence of infarction, or autopsy confirmation.

3.2 Definite haemorrhagic stroke (primary intracerebral, subarachnoid, or secondary to cerebral infarction): confirmation with a CT or MRI scan within 2 weeks of stroke, or at autopsy or by lumbar puncture.

3.3 Stroke of unknown aetiology: definite stroke of unknown aetiology when CT, MRI or autopsy are not done, or where CT or MRI scan does not reveal pathology.

3.4 Non-fatal stroke post cardiovascular invasive interventions: stroke (as defined in 3.1, 3.2 or 3.3) associated to the intervention within 30 days of cardiovascular surgery, or within 7 days of cardiac catheterisation, or angioplasty, atherectomy, stent deployment or other invasive coronary or peripheral vascular interventions.

3.5 Non-fatal stroke post non-cardiovascular surgery: stroke (as defined in 3.1, 3.2 or 3.3) occurring within 30 days of non-cardiovascular surgery.

4.0 Transient ischaemic attacks (TIA)

4.1 Definite TIA: focal neurological deficits with duration of less than 24 hours. Deficits must be observed and described by a physician.

4.2 ProbableTIA: focal neurological deficits with duration of less than 24 hours. Deficits not observed or described by a physician.

4.3 TIA post cardiovascular invasive interventions: TIA (as defined in 4.1 or 4.2) associated to the intervention within 30 days of cardiovascular surgery, or within 7 days of cardiac catheterisation, or angioplasty, atherectomy, stent deployment or other invasive coronary or peripheral vascular interventions.

4.4 TIA post non-cardiovascular surgery: TIA (as defined in 4.1 or 4.2) occurring within 30 days of non-cardiovascular surgery.

5.0 Amputation (includes both amputation and exarticulation)

5.1 Amputation caused by cardiovascular disease

5.2 Amputation caused by pressure wound/gangrene/infection

6.0 Invasive cardiovascular procedures

6.1 Coronar artery bypass graft (CABG)

6.2 Percutaneous transluminal coronary angioplasty (PTCA)

6.3 Attempted PTCA: (to be used in case of an unsuccessful attempt)

6.4 Coronary stent deployment

6.5 Coronary arteriography with stenosis

7.0 Peripheral vascular procedures

7.1 Bypass surgery: state localisation

7.1.1 lower extremity

7.1.2 upper extremity

7.1.3 carotid

7.2 Percutaneous transluminal angioplasty (PTA): state localisation

7.2.1 lower extremity

7.2.2 upper extremity

7.2.3 carotid

7.3 Attempted PTA: (to be used in case of an unsuccessful attempt). state localisation

7.3.1 lower extremity

7.3.2 upper extremity

7.3.3 carotid

7.4 Stent deployment: state localisation

7.4.1 lower extremity

7.4.2 upper extremity

7.4.3 carotid

7.5 Thrombendarterectomy/thrombectomy: state localisation

7.5.1 lower extremity

7.5.2 upper extremity

7.5.3 carotid

7.6 Stenosis in a. carotis: verified by arteriography or Doppler ultrasound

8.0 Death from other than cardiovascular cause

8.1 cancer

8.2 suicide

8.3 hypoglycaemia

8.4 accident

8.5 unspecified

9.0 Ischaemia in ECG

9.1 Ischaemia in resting ECG: Minnesota code 1.1-1.3, 4.1-4.4, 5.1-5.8 or 7.1

9.2 Ischaemia in work load ECG: ST-depression of more than 1 mm in any lead

10.0 Distal blood pressure gradient

Significant decline in distal blood pressure gradient: decline in systolic blood pressure gradient of at least 28 mm Hg between the right arm and great toe in one or both legs.

Apendix 2

Framingham 10 year risk score charts for men and women. Aspirin therapy as primary prevention should be considered of the 10 year risk score exceeds 6% (248). To convert values for cholesterol to millimoles per liter, multiply by 0.02586 Box 1 and Box 2 .