Upsala J Med Sci 105: 2, 135-150,2000 Epidemiological and Clinical Studies on Insulin Resistance and Diabetes Hans Lithell, Johan Sundstrom, Johan Amlov, Kristina Bjorklund, Arvo H h n i , Anu Hedman, Bjorn Zethelius, Liisa Byberg, Lena Kilander, Richard Reneland Department of Public Health and Caring Sciences/Geriatrics, Uppsala University, Uppsala, Sweden ABSTRACT In Uppsala, extensive epidemiological and clinical studies on insulin resistance and diabetes have been ongoing for the past 30 years. A prospective cohort study of men born 1920-24, living in Uppsala County, was initiated during 1969-74 (the Uppsala Longitudinal Study of Adult Men, ULSAM). Risk factors for cardiovascular disease were examined in 2,322 men, and re-examinations have been performed every 10 years. At the first follow-up, when the men were 60 years old, insulin resistance was found to be a risk factor for development of hypertension and diabetes. In addition, treatment with antihypertensive medication was an independent risk factor for development of diabetes. These findings resulted in a series of clinical studies on metabolic effects of antihypertensive agents. At the second follow-up, when the men were 70 years old, the development of hypertension and diabetes was once again in focus, but at this time, cross-sectional and prospective studies of other cardiovascular determinants, such as circadian blood pressure pattern, left ventric- ular geometry and function, muscle morphology, ion status, fibrinolysis and cogni- tive function, were also performed. The cohort has furthermore been linked to the Swedish census and hospital discharge and cause of death registries, it has been used for studies on relationships between birth weight and cardiovascular disease, and genetic analyses have been performed, taking advantage of the long observation time obtained in this cohort. The cohort is currently being re-examined for the third time, and will hopefully continue to provide valuable information on the epidemio- logy of diabetes and cardiovascular disease in the future. INTRODUCTION In the late 1960s, an interest in the pathogenesis for type 2 diabetes mellitus emerged. In Uppsala, a prospective cohort study was planned and executed during 1969-74 (27). It was based on a cohort of 50-year-old men living in Uppsala county, born 1920-24 (the Uppsala Longitudinal Study of Adult Men, ULSAM). Of the 2,841 men available, 2,322 (82%) accepted the invitation to participate in a health 135 survey. A large number of investigations were performed. A questionnaire regarding physical exercise habits, smoking habits and family history of cardiovascular di- sease was filled out by the subjects. Concentrations of fasting lipids, glucose and insulin were measured, and in 1,692 men an intravenous glucose tolerance test (IVGTT) was carried out. At that time there were still no official guidelines on how to investigate glucose intolerance and it was not until 1980 that the first recom- mendations to use oral glucose tolerance tests (OGTT) appeared. However, the use of IVGTT turned out to be fruitful in the epidemiological context. It was also de- cided that follow-up examinations would be performed every 10 years. This is a brief summary of past and ongoing research that has its basis in this cohort study or has been initiated due to findings in the cohort study. FINDINGS AT AGE 60 Insulin resistance and hypertension The first follow-up was carried out in 1980-84, when the men were 60 years old. A total of 1,860 men (87.5% of the eligible men from the original cohort) participated in the follow-up examination. This time the focus was on identifying those men who had developed diabetes or hypertension. For development of hypertension, the blood pressure itself was found to be the most important risk factor. In addition, fasting and late (at 60 minutes of the IVGTT) insulin concentrations, body mass index (BMI), abdominal skinfold thickness and heredity for hypertension were identified as contributing factors. In particular, late insulin concentration and in- crease in BMI were important risk factors for development of hypertension. We believe this was the first prospective study to demonstrate insulin resistance (as reflected in insulin concentrations) being a risk factor for hypertension (66). Insulin resistance and type 2 diabetes mellitus Amongst the 1,834 men who were normoglycemic at age 50 and re-investigated at age 60,77 (4.4%) had developed diabetes at age 60. The most powerful risk factors for development of diabetes were a high fasting insulin concentration (an indicator of insulin resistance), a low insulin response to glucose injection at the IVGTT, BMI, systolic blood pressure and a high glucose value (within the normal range). In addition, treatment with anti-hypertensive medication was an independent risk factor (67). FINDINGS AT AGE 70 Non-dipping and the insulin resistance syndrome Non-dipping is a circadian blood pressure pattern that sometimes accompanies states of autonomic failure (41), diabetes mellitus (1 1) and hypertension (69), but also occurs in apparently healthy subjects. Whether non-dipping adds to an in- creased risk of end organ damage or not has been disputed. A blunted nocturnal 136 blood pressure reduction has been associated with left ventricular hypertrophy (78), renal dysfunction (6), silent cerebrovascular disease (65) and cognitive deterioration (31). In addition, non-dippers have an increased mortality risk compared with dip- pers (50). In contrast, some investigators have failed to find a relationship between an impaired nocturnal blood pressure fall and target organ damage in hypertensive individuals (9, 63). In the ULSAM cohort, 24-hour blood pressure was evaluated in 1,057 70-year-old men. Preliminary data from a cross-sectional investigation in- dicate that non-dippers (n=66), in addition to insulin resistance, diabetes and hyper- triglyceridaemia, have an unfavourable lipid profile with elevated levels of free fatty acids (FFA) in serum and an increased proportion of palmitic, palmitoleic and oleic acid in serum cholesterol esters. Ion status, hypertension and insulin resistance A disturbed magnesium balance, with reduced intracellular ionised magnesium con- centration, has been observed in patients with essential hypertension and in subjects with diabetes mellitus (62). Magnesium supplementation in humans has led to increased insulin sensitivity, lowered blood pressure and improved lipid status (55, 6 1, 80). Increased serum magnesium concentrations during antihypertensive capto- pril treatment were correlated to improved insulin sensitivity (1 8). During bendro- fluazide treatment serum magnesium decreased significantly and there was an inverse correlation between the changes in serum magnesium and mean fasting glucose concentrations (14). An association between the magnesium and calcium balance in skeletal muscle and blood pressure response to antihypertensive treat- ment has been reported (17). The changes in serum magnesium concentrations, and in the ratio between calcium and magnesium concentrations, correlated to changes in insulin sensitivity and to serum triglyceride concentrations during antihyperten- sive treatment with different angiotensin converting enzyme (ACE) inhibitors ( 15, 19). It has been questioned whether the total magnesium concentration in blood reflects the biological activity of magnesium since only about 60 % of the mag- nesium in the blood is in a free unbound state. In fact, atherogenic lipid fractions and some glucometabolic variables were more closely correlated with circulating ionised magnesium than with total magnesium concentration (2 1). The circulating ionised magnesium concentration in patients with essential hypertension and in insulin resistant but otherwise healthy subjects was within the normal range (21). Euglycaemic hyperinsulinaemia increases the circulating ionised magnesium con- centration while no significant change was observed of the total magnesium level (16,20). Relationships between muscle morphology and insulin resistance Skeletal muscle accounts for the largest part of glucose uptake when stimulated by insulin (as much as 90%). The capacity of the muscle to utilise glucose depends largely on muscle structure. Muscle rich in type I (oxidative) fibres, as well as muscle rich in capillaries, apparently show much higher glucose uptake than muscle containing mainly type IIB (glycolytic) fibres. The percentage of type I fibres and 137 capillary density in skeletal muscle increases with increasing level of physical activity. In contrast, type IIB fibres are prevalent in obese people. Muscle biopsies were taken in 515 men (43%) at age 70 in order to analyse muscle histology. Results of this analysis showed a clear association between insulin sensi- tivity and muscle structure. Insulin sensitivity showed a positive relationship with percentage of type I fibres and capillary density and a negative correlation with per- centage of type IIB fibres (25). These associations were independent of the level of physical activity and obesity. Moreover, diabetic subjects showed a significantly higher percentage of type IIB muscle fibres compared to healthy controls. A more intriguing and less studied aspect is the degree to which muscle structure, i.e. fibre composition and capillary density, is related to hypertension. For that pur- pose, a group of normotensive (n=113) and hypertensive (n=43) men were com- pared in order to test the hypothesis of a relationship between heart rate, develop- ment of hypertension and muscle morphology in a state of normal glucose tole- rance. These groups did not differ significantly with regard to fibre type distribu- tion, but the hypertensive subjects had a significantly lower capillary supply than the controls, when analysed as number of capillaries around different fibre types. Capillary rarefaction in the hypertensive subjects in the present study was closely associated with the increase in blood pressure over a 20-year period. Heart rate, a marker of the balance between sympathetic and parasympathetic tone, was inverse- ly correlated to capillary supply, which supports the hypothesis of involvement of increased sympathetic tone in a reduction of the capillary net in skeletal muscle (26). Insulin has also been found to increase the blood flow in skeletal muscle beds through vasodilation. This increase in blood flow is believed to contribute to glucose uptake by the muscle. The magnitude of the increase in blood flow during hyperinsulinaemia, which is endothelium-dependent, depends not only on the degree of vasodilation in large vessels, but also possibly on the number of capil- laries present in the muscle bed. Preliminary data show that the capillary density is associated to insulin-induced changes in leg blood flow. This indicates that endo- thelial function is closely linked to the capillarisation in skeletal muscle. Proinsulin in relation to development of type 2 diabetes and cardiovascular disease In longitudinal studies, the fasting concentration of total proinsulin-like molecules predicts the development of type 2 diabetes over periods of follow-up ranging from 2 to 4 years (76). Fasting concentrations of specific insulin, intact and 32-33 split proinsulin have been measured in plasma samples, stored frozen since baseline in liquid nitrogen, by the specific two-site immunometric assay technique (68). Zethe- lius and co-workers have published preliminary results (83) indicating that proinsu- lin may be a long-term predictor for diabetes when compared to known baseline risk factors for type 2 diabetes in this cohort (glucose and immunoreactive insulin concentrations, fasting and at 60 min after an intravenous glucose load, respectively, and use of antihypertensive drug treatment). 138 Insulin has been shown to be a risk factor for cardiovascular disease (64). The extent to which proinsulin may contribute to the association between insulin and cardiovascular disease is largely unknown. The aim of one of our studies was to identify the longitudinal relationship between proinsulin and coronary heart disease in the ULSAM cohort. The diagnosis of incident disease was collected from the official in-patient and causes-of-death registers. In a preliminary report (82), Zethe- lius and co-workers have shown results indicating that proinsulin is a predictor for cardiovascular disease and may be independent of known confounders. Heart failure and the insulin resistance syndrome Heart failure is the only major cardiovascular disorder that is increasing in in- cidence and prevalence (44), with substantial morbidity and mortality and a huge economic impact on the public health systems of western society (28). In spite of this, the epidemiological data on heart failure are relatively scarce. Previous studies demonstrate an association between insulin resistance and heart failure (53, 74) and an impaired insulin-mediated glucose uptake has been shown to be a prognostic fac- tor in heart failure patients (56). Preliminary results from h l o v and co-workers (84) indicate that factors related to insulin resistance (10, 49, 52, 79), such as an increased heart rate, increased serum concentrations of proinsulin, a high proportion of dihomogammalinolenic acid in serum cholesterol esters and hypophosphataemia are predictors of left ventricular systolic dysfunction in elderly males after 20 years follow-up, independently of myocardial infarction, hypertension, diabetes and the use of cardiovascular medication. Left ventricular hypertrophy and the insulin resistance syndrome Left ventricular hypertrophy (LVH) is an important risk factor for cardiovascular and all-cause mortality and morbidity (32, 37), and an increased left ventricular relative wall thickness (RWT, wall thickness divided by ventricular diameter) also has an adverse prognosis (32,77). The aetiology of LVH is largely unknown. Cross- sectional studies (23, 36, 42, 60, 70) have shown that male sex, age, hypertension, obesity, valvular disease, previous myocardial infarction, heredity and alcohol use are important in LVH. Recently, associations between LVH and the insulin resist- ance syndrome have been found (39, 43, 54), although some studies (51, 71) have found insulin resistance or impaired glucose tolerance to be more closely related to increased RWT than to LVH. In the ULSAM cohort, electrocardiographic and echo- cardiographic examinations were performed in 583 men at 70 years of age. Thus, this cohort is well suited for the much needed further study of the epidemiology of LVH, including detailed cross-sectional and prospective studies of the relationship between aspects of the insulin resistance syndrome and left ventricular geometry, as well as development of new diagnostic electrocardiographic criteria for LVH. In a cross-sectional study of the cohort at age 70 (7 l), we found that several com- ponents of the insulin resistance syndrome (hyperinsulinaemic euglycaemic clamp insulin sensitivity index, fasting insulin, 32-33 split proinsulin, triglycerides, FFA, OG'IT glucose and insulin levels, waisthip ratio, BMI, 24-h blood pressure and 139 heart rate) were related to left ventricular RWT. Only 24-h systolic pressure and heart rate were related to left ventricular mass index. Several metabolic parameters were adversely altered in left ventricular concentric remodelling, but only 24-h blood pressure and heart rate in LVH. Preliminary data from a small study using positron emission tomography to determine myocardial, skeletal muscle and whole-body insulin sensitivity indicate that myocardial insulin sensitivity may be related to RWT and not to left ventricular mass index (73). Preliminary studies of predictors for LVH over 20 years indicate that dyslipidaemia and indices of a low dietary intake of linoleic acid and high intake of saturated and monounsaturated fats, as well as hypertension and obesity, at age 50 are predictive of the prevalence of LVH 20 years later, suggesting that lipids may be important in the aetiology of LVH (72). Insulin resistance and PAI-I Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are regarded as a com- ponent of the insulin resistance syndrome (1). PAI-1 is an important factor in the regulation of fibrinolysis and inhibits the degradation of solid fibrin by forming an inactive complex with tissue-type plasminogen activator (t-PA). Plasminogen is cleaved by t-PA to form active plasmin that degrades fibrin clots. It has been suggested that PAI-1 is one of the factors linking insulin resistance with cardiovas- cular disease (29). PAI-1 activity was analysed in the ULSAM cohort at age 70 (8). BMI, waisthip ratio, blood pressure, fasting glucose, insulin and serum triglyceride concentrations were all positively related with PAI- 1 activity. Insulin sensitivity index, measured by clamp, and HDL cholesterol concentrations were inversely related with PAI- 1 ac- tivity. In a multivariate regression analysis, BMI, waisthip ratio, serum triglyceride concentrations, and insulin sensitivity were related to the PAI- 1 activity, all indepen- dent of each other. PAI-1 activity was also higher in men with type 2 diabetes and impaired glucose tolerance than in men with normal glucose tolerance. This dif- ference was not explained by variations in BMI, waisthip ratio, serum triglycerides, insulin sensitivity, blood pressure medication or fasting glucose concentrations. Insulin resistance and cognitive function In recent years, it has been recognised that cerebrovascular disease is associated not only with stroke and vascular dementia, but also with late-onset Alzheimer’s di- sease. Dementia research has now focused on risk factors associated with early stages of cognitive impairment, from which a further progress to dementia may be prevented. A subgroup of the 70-year-old men (999 out of 1,221) participated in cognitive function testing. The Mini Mental State Examination (12) and the Trail Making Tests (38) were used, tests which are sensitive for subcortico-frontal dys- function. Results have been previously published in detail (30, 31). In brief, men with diabetes according to an OGTT (n=130) had significantly poorer results on the cognitive tests, even when stroke, hypertension and atrial fibrillation were taken into account. Similarly, clamp measurements of insulin sensitivity were related to 140 cognitive function: men in the highest tertile of insulin sensitivity performed sig- nificantly better than those in the lowest tertile. STUDIES ON LOW BIRTH WEIGHT, INSULIN RESISTANCE AND DIABETES Low birth weight predicts physiological disturbances in adult life, such as raised blood pressure (3, 33, 34), type 2 diabetes (22, 40, 4 3 , impaired glucose tolerance (22, 59) and insulin resistance (40, 46, 58), all constituents of the insulin resistance syndrome. All of these conditions are associated with an increased risk of and mortality from cardiovascular disease, as is small size at birth (5, 35). Low birth weight has also been associated with the insulin resistance syndrome itself (4, 75). Birth weight, in this cohort, is inversely associated with fasting concentrations of immunoreactive insulin and insulin resistance at age 50 and positively associated with BMI at age 50 (40) and insulin sensitivity at age 70 (46). Birth weight was also related to blood pressure at both ages (33, 34) but not with the concentrations of serum triglycerides or HDL cholesterol at age 50 (40). In a recent study we demon- strated that low birth weight is associated with physiological disturbances in adult age that are a subset of the disturbances clustered in the general population as the insulin resistance syndrome (7). Birth weight was inversely associated with the subscapular/triceps skinfold ratio investigated at age 50 and used as a measure of truncal fat. Low birth weight predicted a smaller hip but not a larger waist and thus the inverse relationship of birth weight with the waisthip ratio at age 70 does not reflect an association of low birth weight with central adiposity. Birth weight was also inversely associated with the activity of PAI-1 and the concentrations of specific insulin and proinsulin at age 70. There were no associations of birth weight with the concentrations of serum triglycerides or HDL cholesterol at age 70. In short, low birth weight predicts high blood pressure, insulin resistance, truncal obesity and high PAI-1 activity but not the abdominal obesity or dyslipidaemia present in the insulin resistance syndrome. Understanding why low birth weight predicts only some components of the insulin resistance syndrome and not others may depend on identifying a specific physiological pathway that links this subset of disturbances. One possible mechanism could be increased concentrations of or sensitivity to glucocorticoids. Supporting this hypothesis are the observations that long-term treatment of glucocorticoids cause accumulation of truncal fat, gluco- corticoids increase the production of PAL1 (48), and the sensitivity to glucocorti- coids could be programmed in foetal life (57). EFFECTS OF ANTIHYPERTENSIVE DRUGS ON INSULIN RESISTANCE In eight different intervention studies, the actively treated comparison groups have been treated with a selective beta-blocker (atenolol). The average effect on insulin sensitivity was a reduction by 19% from the basal value after 4-6 weeks placebo 141 treatment. The average effect of two similar diuretics, given in moderate high doses, was a decrease by 19% in insulin sensitivity. In four studies of calcium-channel blockers, no significant effect on insulin sensitivity was found. In the class of ACE inhibitors, the results ranged from +16% with captopril to -10% with lisinopril. The average effect was +2%. With two different angiotensin,-receptor blockers, similar non-significant (+2%) effects on insulin sensitivity were demonstrated. The average of three different studies with alpha-1 selective inhibitors was an improvement of insulin sensitivity by 23%. The effect of moxonidine treatment was an improvement of insulin sensitivity by 12%. However, the improvement was restricted to a sub- group of very insulin resistant patients, predefined in the protocol, who improved by 21%. Moxonidine belongs to a class of centrally acting inhibitors of the sympa- thetic nervous system. The agonistic effect these drugs exhibit on the imidazoline I, receptors causes an inhibition of the sympathetic nervous system. CANDIDATE GENES FOR INSULIN RESISTANCE For several years, the Geriatrics Section of the Department of Public Health and Caring Sciences has actively collaborated with Professor Oluf Pedersen’s research group at Steno Diabetes Centre in Gentofte, Denmark. The Steno group investigates candidate genes for type 2 diabetes. Variants of candidate genes are identified either from the literature or in-house at Steno. Associations between these genetic variants and relevant phenotypes such as type 2 diabetes, insulin resistance, and insulin secretion are then sought in the ULSAM material. In ULSAM, DNA has been collected from >95% of participants and IVGTT, OGTT and hyperinsulinaemic, euglycaemic clamp data are available. In the first study, hepatocyte nuclear factor 4alpha (HNF-4a), the MODY- 1 gene, was investigated, and two single nucleotide polymorphisms that changed the amino acid sequence were found, one previously reported (Thrl30Ile) and one new (Va1255Met). In a Danish sample, the frequency of the Thrl30Ile variant was sig- nificantly higher in 509 type 2 diabetes patients (4.7%) compared to control sub- jects (1.7%, p=0.008). This finding could not be replicated in the Swedish ULSAM population, however, in which frequencies of the variant were similar in subjects with type 2 diabetes (5.4%) and glucose tolerant control subjects (5.1 %). The novel Va1255Met variant was originally only found in type 2 diabetes patients (4/477) and not in controls (0/217). Interestingly, the Va1255Met variant could not be found in any of the 894 ULSAM men investigated (47). In a second study, the previously identified Gly1057Asp variant of the insulin receptor substrate-2 (IRS-2) gene was analysed in four different groups of glucose tolerant subjects, one of which was derived from ULSAM. No consistent effect on insulin sensitivity or secretion was found (2). A third study investigated the influence of two common genetic variants in the PPPlR3 gene encoding the glycogen targeting subunit of type-1 protein phos- phatase (24). A previous study in Pima Indians indicated an association between the Asp905Tyr, Arg883Ser, or PPl ARE polymorphisms, and insulin sensitivity (8 1). 142 The Arg883Ser variant was not included in the DanisWSwedish study since it could not be found in 82 Caucasian volunteers. In ULSAM there was no association be- tween either the Asp905Tyr or the PPlARE variants and diabetes incidence be- tween 50 and 70 years of age. There was no association between the Asp905Tyr variant and insulin sensitivity, but there was a weak correlation between the PPlARE variant and whole body insulin sensitivity. In view of the multiple tests involved in the study, however, this correlation was not considered significant by the authors (24). DISCUSSION The past and on-going research of our group reviewed in this paper demonstrates the usefulness of an epidemiological study as a basis for research. Not only can valuable information be obtained from the study itself, but other studies may de- velop from some of the, often unexpected, results. In this case, some of the findings resulted in a series of clinical studies on metabolic effects of antihypertensive agents. In addition, the cohort was useful for studies on relationships between birth weight and cardiovascular disease, and for genetic analyses. In the first follow-up of the ULSAM cohort (at age 60), two major findings were made. Development of hypertension at age 60 had been preceded by high insulin concentrations at age 50. Insulin resistance as a predictor of hypertension has been found in some but not all other studies, and the question of whether insulin is causally related to hypertension is still debated. The other major finding was that high fasting insulin concentrations (as indicators of insulin resistance) and low in- sulin response to glucose injection at the IVG'IT (as a measure of insulin secretion capacity) were strong predictors of future diabetes. The importance of insulin re- sistance for diabetes was further manifested in the 20-year follow-up. We used stored, frozen serum samples and analysed for specific insulin, proinsulin and 32- 33 split proinsulin. Proinsulin (which reflects insulin resistance) was a powerful risk factor for development of diabetes, particularly during the first ten years. At the second follow-up (at age 70), skeletal muscle biopsies were studied in about 500 of the participants. Investigations of muscle fibre composition and capil- larisation indicated that hypertensive, glucose-tolerant 70-year-old men have a lower percentage of type I fibres and more type I1 fibres than a control group of normotensive men. Furthermore, the capillarisation was less pronounced. Capillari- sation was related to insulin sensitivity and to blood flow changes during hyperin- sulinaemia, and FFA concentrations also played an important role in determining the vasodilatory response to hyperinsulinaemia. We demonstrated a relationship be- tween heart rate at age 50 and low capillary density at age 70 as well as between heart rate at 50 and development of hypertension during the following 20 years. This indicates a possible influence of high sympathetic tone being involved in both structural changes in skeletal muscle and hypertension. Extensive echocardiographic investigations were performed in nearly 600 men. These data have now been studied with regard to metabolic characteristics of dif- 143 ferent morphologic measures. Several characteristics of the metabolic syndrome and insulin resistance itself Correlated to the left ventricular relative wall thickness, in contrast to the findings for left ventricular hypertrophy, with which only 24-h blood pressure and heart rate were correlated. Skeletal muscle insulin resistance may thus be important for development of increased relative wall thickness, as supported from the study with positron emission tomography. In this study population, it is interesting that insulin resistance and proinsulin concentrations are significant predictors also for coronary heart disease and left ventricular systolic dysfunction. Some of the effect on coronary heart disease may be mediated by PAI-1 activity, which is strongly correlated to insulin resistance. A deleterious effect of insulin resistance also on small vessels may explain the correla- tion between cognitive function and insulin resistance/diabetes mellitus. The metabolic consequences of antihypertensive medication have been exten- sively described in the original publications and will not be commented in detail here. The increased insulin resistance of beta-blockers and diuretics most likely explains the increased risk for diabetes mellitus associated with use of these drugs, as high doses were used in the 1970s. This was recently confirmed in a large study for beta-blockers but not for diuretics (1 3). The doses used in the study are not provided in the report, but it seems likely that they were lower for diuretics in the study than in the 1970s. Much of our data (in co-operation with the London School of Hygiene and Tropical Medicine, London, UK) on the relationship between low birth weight and risk for diabetes confirms David Barker’s findings (3-5). However, we have demon- strated that this increased risk is mediated by an effect of birth weight on insulin resistance and not on insulin secretion. In our population, it seems that only part of the syndrome is related to birth weight, in particular those metabolic variables that are related to cortisol effects. The effect of candidate genes for insulin resistance has been studied in this cohort in co-operation with Steno Diabetes Centre in Copenhagen. This project is ongoing. The ULSAM population supplements some of the younger populations of Cau- casian origin in the Steno studies. In summary, these studies on insulin resistance and diabetes are based on a pro- spective cohort study of medium size. The population is small for genetic studies, but can supplement studies in younger populations in order to describe the different gene-environment interactions at different ages. Most of the findings described here are the preliminary results for some of our Ph.D. students, whose dissertations will soon be providing more in-depth analysis of the ULSAM data. A list of publications and more detailed information on the cohort can be found at the web-site www.pub- care.uu.se/ulsam. REFERENCES 1 . Alberti, K. G . M. M. & Zimmet, P. Z. Definition, diagnosis and classification of dia- betes mellitus and its complications. Part l : Diagnosis and classification of diabetes 144 mellitus. Provisional report of a WHO consultation. In: for the WHO Consultation. Dia- bet Med; 1998. p. 539-553. 2. Almind, K., Frederiksen, S. K., Bernal, D., Hansen, T., Ambye, L., Urhammer, S., et al. Search for variants of the gene-promoter and the potential phosphotyrosine encoding sequence of the insulin receptor substrate-2 gene: evaluation of their relation with al- terations in insulin secretion and insulin sensitivity. Diabetologia 42: 1244-1 249, 1999. 3. Barker, D. J. P., Bull, A. R., Osmond, C. & Simmonds, S. J. Fetal and placental size and risk of hypertension in adult life. BMJ 301:259-262, 1990. 4. Barker, D. J. P., Hales, C. N., Fall, C. H. D., Osmond, C., Phipps, K. & Clark, P. M. S. Type 2 (non-insulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relation to reduced fetal growth. Diabetologia 36:62-67, 1993. 5. Barker, D. J. P., Osmond, C., Simmonds, S. J. & Wield, G. A. The relation of small head circumference and thinness at birth to death from cardiovascular disease in adult life. BMJ 306:422426, 1993. 6. Bianchi, S., Bigazzi, R., Baldari, G., Sgherri, G. & Campese, V. M. Diurnal variations of blood pressure and microalbuminuria in essential hypertension. Am J Hypertens 7. Byberg, L., McKeigue, P. M., Zethelius, B. & Lithell, H. 0. Birth weight and the in- sulin resistance syndrome: association of low birth weight with truncal obesity and raised plasminogen activator inhibitor- 1 but not with abdominal obesity or plasma lipid disturbances. Diabetologia 43:54-60,2000. 8. Byberg, L., Siegbahn, A., Berglund, L., McKeigue, P., Reneland, R. & Lithell, H. Plas- minogen activator inhibitor- 1 activity is independently related to both insulin sensitivity and serum triglycerides in 70-year-old men. Arterioscler Thromb Vasc Biol 18:258- 264, 1998. 9. Cuspidi, C., Lonati, L., Sampieri, L., Macca, G., Valagussa, L., Zaro, T., et al. Impact of nocturnal fall in blood pressure on early cardiovascular changes in essential hyper- tension. J Hypertens 17:1339-1344, 1999. 10. DeFronzo, R.A. & Lang, R. Hypophosphatemia and glucose intolerance: evidence for tissue insensitivity to insulin. N Engl J Med 303:1259-1263, 1980. 11. Fogari, R., Zoppi, A., Malamani, G.D., Lazzari, P., Destro, M. & Corradi, L. Ambu- latory blood pressure monitoring in normotensive and hypertensive type 2 diabetes. Prevalence of impaired diurnal blood pressure patterns. Am J Hypertens 6:l-7, 1993. 12. Folstein, M. F., Folstein, S. E. & McHugh, P. R. “Mini-mental state”. A practical meth- od for grading the cognitive state of patients for the clinician. J Psychiatr Res 12: 189- 198, 1975. 13. Gress, T. W., Nieto, F. J., Shahar, E., Wofford, M. R. & Brancati, F. L. Hypertension and antihypertensive therapy as risk factors for type 2 diabetes mellitus. Atherosclerosis Risk in Communities Study. N Engl J Med 342:905-912,2OOO. 14. Haenni, A., Andersson, P. E., Lind, L. et al. Electrolyte changes and metabolic effects of lisinoprilhendrofluazide treatment. Am J Hypertens 7:615-622, 1994. 15. Haenni, A., Berglund, L., Reneland, R. et al. The alterations in insulin sensitivity during ACE inhibitor treatment are related to calciudmagnesium balance. Am J Hypertens 16. Haenni, A., Lind, L., Andersson, A. et al. Ionized magnesium status during hyperin- sulinemia in hypertension. In: Kiss, editor. 6th European Magnesium Congress; 1998; Budapest: Hungarian Magnesium Society; 1998. p. 57. 17. Haenni, A., Lind, L. & Lithell, H. The blood pressure response to antihypertensive 7~23-29, 1994. 10: 145-1 5 I , 1997. 145 treatment with lisinopril or bendrofluazide is related to the calcium and magnesium contents in skeletal muscle. Am J Hypertens 9:273-276, 1996. 18. Haenni, A., Lind, L. & Lithell, H. The improved glucose metabolism of captopril treat- ment is related to altered magnesium status. J Hypertens 12:S49, 1994. 19. Haenni, A., Reneland, R., Anderson, P.E. et al. The alterations in insulin sensitivity during ACE inhibitor treatment are related to magnesium balance. Magnesium Res 1:190-196, 1997. 20. Haenni, A., Reneland, R., Lind, L. et al. Increased serum aldosterone during hyperinsu- linemia in obese insulin resistant patients with essential hypertension. J Hypertens, 2000. 2 1. Haenni, A., Ohrvall, M. & Lithell, H. Atherogenic lipid fractions are related to ionized magnesium status. Am J Clin Nutr 67:202-207, 1998. 22. Hales, C. N., Barker, D. J. P., Clark, P. M. S., Cox, L. J., Fall, C., Osmond, C., et al. Fetal and infant growth and impaired glucose tolerance at age 64. BMJ 303:1019-1022, 1991. 23. Hammond, I.W., Devereux, R.B., Alderman, M.H. & Laragh, J.H. Relation of blood pressure and body build to left ventricular mass in normotensive and hypertensive employed adults. J Am Coll Cardiol 12:996-1004, 1988. 24. Hansen, L. et al. Polymorphism in the glycogen-associated regulatory subunit of type 1 protein phosphatase (PPPlR3) gene and insulin sensitivity. Diabetes 49:298-301,2000. 25. Hedman, A., Berglund, L., EssCn-Gustavsson, B., Reneland, R. & Lithell, H. Relation- ships between muscle morphology and insulin sensitivity are improved after adjustment for intraindividual variability in 70-year-old-men. Acta Physiol Scand 168:in press, 2000. 26. Hedman, A., Reneland, R. & Lithell, H. Alterations in skeletal muscle morphology in glucose tolerant elderly hypertensive men: relationship to development of hypertension and heart rate. J Hypertension 18559-565,2000. 27. Hedstrand, H. A study of middle-aged men with particular reference to risk factors for cardiovascular disease. Dissertation. Ups J Med Sci suppl 19, 1975. 28. Hunink, M. G., Goldman, L., Tosteson, A. N., Mittleman, M. A., Goldman, P. A., Wil- liams, L. W., et al. The recent decline in mortality from coronary heart disease, 1980- 1990. The effect of secular trends in risk factors and treatment. Jama 277535-542, 1997. 29. Juhan-Vague, I., Alessi, M.C. & Vague, P. Increased plasma plasminogen activator inhibitor 1 levels. A possible link between insulin resistance and atherothrombosis. Diabetologia 34:457462, 1991. 30. Kilander, L., Andren, B., Nyman, H., Lind, L., Boberg, M. & Lithell, H. Atrial fibrilla- tion is an independent determinant of low cognitive function: a cross-sectional study in elderly men. Stroke 29:1816-1820, 1998. 3 1. Kilander, L., Nyman, H., Boberg, M., Hansson, L. & Lithell, H. Hypertension is related to cognitive impairment: a 20-year follow-up of 999 men. Hypertension 31:780-786, 1998. 32. Koren, M. J., Devereux, R. B., Casale, P. N., Savage, D. D. & Laragh, J. H. Relation of left ventricular mass and geometry to morbidity and mortality in uncomplicated essential hypertension. Ann Intern Med 114:345-352, 1991. 33. Koupilovh, I., Leon, D. A., Lithell, H. 0. & Berglund, L. Size at birth and hypertension in longitudinally followed 50-70-year-old men. Blood Press 6:223-228, 1997. 34. Leon, D. A., Koupilova, I., Lithell, H. O., Berglund, L., Mohsen, R., Vigero, D., et al. 146 Failure to realise growth potential in utero and adult obesity in relation to blood pres- sure in 50 year old Swedish men. BMJ 312:401406, 1996. 35. Leon, D. A., Lithell, H. O., Vigero, D., KoupilovB, I., Mohsen, R., Berglund, L., et al. Reduced fetal growth rate and increased risk of death from ischaemic heart disease: cohort study of 15 000 Swedish men and women born 1915-29. BMJ 317:241-245, 1998. 36. Levy, D., Anderson, K. M., Savage, D. D., Kannel, W. B., Christiansen, J. C. & Castelli, W. P. Echocardiographically detected left ventricular hypertrophy: prevalence and risk factors. The Framingham Heart Study. Ann Intern Med 108:7-13, 1988. 37. Levy, D., Garrison, R. J., Savage, D. D., Kannel, W. B. & Castelli, W. P. Prognostic implications of echocardiographically determined left ventricular mass in the Framing- ham Heart Study. N Engl J Med 322:1561-1566, 1990. 38. Lezak, M. Neuropsychological assessment. 3 ed. New York: Oxford University Press; 1995. 39. Lind, L., Anderson, P. E., Andren, B., Hanni, A. & Lithell, H. 0. Left ventricular hypertrophy in hypertension is associated with the insulin resistance metabolic syn- drome. J Hypertens 13:433438,1995. 40. Lithell, H. O., McKeigue, P. M., Berglund, L., Mohsen, R., Lithell, U.-B. & Leon, D. A. Relation of size at birth to non-insulin-dependent diabetes and insulin concentrations in men aged 50-60 years. BMJ 312:406410, 1996. 41. Mann, S., Altman, D. G., Raftery, E. B. & Bannister, R. Circadian variation of blood pressure in autonomic failure. Circulation 68:477483, 1983. 42. Manolio, T. A., Levy, D., Garrison, R. J., Castelli, W. P. & Kannel, W. B. Relation of alcohol intake to left ventricular mass: The Framingham Study. J Am Coll Cardiol 43. Marcus, R., Krause, L., Weder, A. B., Dominguez-Meja, A., Schork, N.J. & Julius, S. Sex-specific determinants of increased left ventricular mass in the Tecumseh Blood Pressure Study. Circulation 90:928-936, 1994. 44. Massie, B. M. & Shah, N. B. Evolving trends in the epidemiologic factors of heart failure: rationale for preventive strategies and comprehensive disease management. Am Heart J 133:703-712,1997. 45. McCance, D. R., Pettitt, D. J., Hanson, R. L., Jacobsson, L. T., Knowler, W. C. & Ben- nett, P. H. Birth weight and non-insulin dependent diabetes: thrifty genotype, thrifty phenotype, or surviving small baby genotype? BMJ 308:942-945,1994. 46. McKeigue, P. M., Lithell, H. 0. & Leon, D. A. Glucose tolerance and resistance to insulin-stimulated glucose uptake in men aged 70 years in relation to size at birth. Dia- betologia41:1133-1138, 1998. 47. Moller, A. M., Urhammer, S. A., Dalgaard, L. T., Reneland, R., Berglund, L., Hansen, T., et al. Studies of the genetic variability of the coding region of the hepatocyte nuclear factor-4alpha in Caucasians with maturity onset NIDDM. Diabetologia 40:980-983, 1997. 48. Morange, P. E., Aubert, J., Peiretti, F., Lijnen, H. R., Vague, P., Verdier, M., et al. Glu- cocorticoids and insulin promote plasminogen activator inhibitor 1 production by human adipose tissue. Diabetes 48:890-895, 1999. 49. Mykkanen, L., Haffner, S.M., Hales, C.N., Ronnemaa, T. & Laakso, M. The relation of proinsulin, insulin, and proinsulin-to-insulin ratio to insulin sensitivity and acute insulin response in normoglycemic subjects. Diabetes 46: 1990-1995, 1997. 50. Ohkubo, T., Imai, Y., Tsuji, I., Nagai, K., Watanabe, N., Minami, N., et al. Relation 17:7 17-721, 1991. 147 between nocturnal decline in blood pressure and mortality. The Ohasama Study. Am J Hypertens 10:1201-1207, 1997. 51. Ohya, Y., Abe, I., Fujii, K., Ohmori, S., Onaka, U., Kobayashi, K., et al. Hyperinsuline- mia and left ventricular geometry in a work-site population in Japan. Hypertension 52. Palatini, P. & Julius, S. Association of tachycardia with morbidity and mortality: patho- physiological considerations. J Hum Hypertens 1 1 Suppl 1 :S 19-27, 1997. 53. Paolisso, G., De Riu, S., Marrazzo, G., Verza, M., Varricchio, M. & D’Onofrio, F. Insulin resistance and hyperinsulinemia in patients with chronic congestive heart failure. Metabolism 40:972-977, 1991. 54. Paolisso, G., Galderisi, M., Tagliamonte, M.R., de Divitis, M., Galzerano, D., Petrocelli, A., et al. Myocardial wall thickness and left ventricular geometry in hypertensives. Relationship with insulin. Am J Hypertens 10: 1250-1256, 1997. 55. Paolisso, G., Sgambato, S., Pizza, G. et al. Improved insulin response and action by chronic magnesium administration in aged NIDDM subjects. Diabetes Care 12:265- 269, 1989. 56. Paolisso, G., Tagliamonte, M. R., Rizzo, M. R., Gambardella, A., Gualdiero, P., Lama, D., et al. Prognostic importance of insulin-mediated glucose uptake in aged patients with congestive heart failure secondary to mitral and/or aortic valve disease. Am J Cardiol 83:1338-1344, 1999. 57. Phillips, D. I. W. Birth weight and the future development of diabetes. A review of the evidence. Diabetes Care 21:B150-B155, 1998. 58. Phillips, D. I. W., Barker, D. J. P., Hales, C. N., Hirst, S. & Osmond, C. Thinness at birth and insulin resistance in adult life. Diabetologia 37: 150-154, 1994. 59. Phillips, D. I. W., Hirst, S., Clark, P. M. S., Hales, C. N. & Osmond, C. Fetal growth and insulin secretion in adult life. Diabetologia 37592-596, 1994. 60. Post, W. S., Larson, M. G., Myers, R. H., Galderisi, M. & Levy, D. Heritability of left ventricular mass: the Framingham Heart Study. Hypertension 30: 1025-1028, 1997. 61. Rasmussen, H. S., Aurup, P., Goldstein, K. & al, e. Influence of magnesium substitu- tion therapy on blood lipid composition in patients with ischemic heart disease. Arch Intern Med 149:105&1053, 1989. 62. Resnick, L. M., Gupta, R. K., Bhargava, K. K. & al, e. Cellular ions in hypertension, diabetes, and obesity. Hypertension 17:95 1-957, 199 1. 63. Roman, M. J., Pickering, T. G., Schwartz, J. E., Cavallini, M. C., Pini, R. & Devereux, R. B. Is the absence of a normal nocturnal fall in blood pressure (nondipping) associ- ated with cardiovascular target organ damage? J Hypertens 15:969-978, 1997. 64. Ruige, J. B., Assendelft, W. J., Dekker, J. M., Kostense, P.J., Heine, R.J. & Bouter, L. M. Insulin and risk of cardiovascular disease: a meta-analysis. Circulation 97:996- 1001, 1998. 65. Shimada, K., Kawamoto, A., Matsubayashi, K., Nishinaga, M., Kimura, S. & Ozawa, T. Diurnal blood pressure variations and silent cerebrovascular damage in elderly patients with hypertension. J Hypertens 10:875-878, 1992. 66. Skarfors, E. T., Lithell, H. 0. & Selinus, I. Risk factors for the development of hyper- tension: a 10-year longitudinal study in middle-aged men. J Hypertens 9:217-223, 1991. 67. Skarfors, E. T., Selinus, K. I. & Lithell, H. 0. Risk factors for developing non-in- sulin dependent diabetes: a 10 year follow up of men in Uppsala. Bmj 303:755-760, 1991. 27:729-734, 1996. 148 68. Sobey, W. J., Beer, S. F., Carrington, C. A., Clark, P. M., Frank, B. H., Gray, I. P., et al. Sensitive and specific two-site immunoradiometric assays for human insulin, proinsu- lin, 65-66 split and 32-33 split proinsulins. Biochem J 260535-541, 1989. 69. Staessen, J. A., Bieniaszewski, L., E, 0. B., Gosse, P., Hayashi, H., Imai, Y., et al. Noc- turnal blood pressure fall on ambulatory monitoring in a large international database. The “Ad Hoc” Working Group. Hypertension 29:30-39, 1997. 70. Sundstrom, J., Lind, L., Andren, B. & Lithell, H. Left ventricular geometry and func- tion are related to electrocardiographic characteristics and diagnoses. Clin Physiol 18:463470, 1998. 71. Sundstrom, J., Lind, L., Nystrom, N., Zethelius, B., Andren, B., Hales, C. N., et al. Left ventricular concentric remodeling rather than left ventricular hypertrophy is related to the insulin resistance syndrome in elderly Men. Circulation 101 :2595-2600,2000. 72. Sundstrom, J., Lind, L., Vessby, B., Andren, B., Aro, A. & Lithell, H. 0. Dyslipidemia and an unfavorable fatty acid profile predict left ventricular hypertrophy twenty years later. Eur J Endocrinol 142 [suppl 1]:17 (abstract), 2000. 73. Sundstrom, J., Lind, L., Valind, S., Waldenstrom, A., Bjomtorp, P. & Lithell, H. 0. Insulin-mediated glucose uptake is not altered in the myocardium in normotensive elderly males with left ventricular hypertrophy. J Hypertension 17 [Suppl 3]:S139 (abstract), 1999. 74. Swan, J. W., Anker, S. D., Walton, C., Godsland, I. F., Clark, A.L., Leyva, F., et al. Insulin resistance in chronic heart failure: relation to severity and etiology of heart fai- lure. J Am Coll Cardiol 30527-532, 1997. 75. Valdez, R., Athens, M. A., Thompson, G. H., Bradshaw, B. S. & Stern, M. P. Birth- weight and adult health outcomes in a biethnic population in the USA. Diabetologia 37:624-631, 1994. 76. Wareham, N. J., Byme, C. D., Williams, R., Day, N. E. & Hales, C. N. Fasting proinsu- lin concentrations predict the development of type 2 diabetes. Diabetes Care 22:262- 270, 1999. 77. Verdecchia, P., Schillaci, G., Borgioni, C., Ciucci, A., Battistelli, M., Bartoccini, C., et al. Adverse prognostic significance of concentric remodeling of the left ventricle in hypertensive patients with normal left ventricular mass. J Am Coll Cardiol 2 5 9 7 1-878, 1995. 78. Verdecchia, P., Schillaci, G., Guerrieri, M., Gatteschi, C., Benemio, G., Boldrini, F., et al. Circadian blood pressure changes and left ventricular hypertrophy in essential hypertension [see comments]. Circulation 8 1528-536, 1990. 79. Vessby, B., Tengblad, S. & Lithell, H. Insulin sensitivity is related to the fatty acid composition of serum lipids and skeletal muscle phospholipids in 70-year-old men. Diabetologia 37: 1044-1050, 1994. 80. Widman, L., Wester, P. O., Stegmayr, B. K. et al. The dose-dependent reduction in blood pressure through administration of magnesium. Am J Hypertens 6:4145, 1993. 81. Xia, J., Scherer, S. W., Cohen, P. T., Majer, M., Xi, T., Norman, R. A., et al. A com- mon variant in PPPIR3 associated with insulin resistance and type 2 diabetes. Diabetes 82. Zethelius, B., Byberg, L., Lithell, H., Hales, N. C. & Berne, C. Proisulin, a Risk factor for Cardiovascular Disease. Diabetologia 42 [suppl 1]:A5: 10 (abstract), 1999. 83. Zethelius, B., Lithell, H., Hales, N. C. & Berne, C. Proinsulin as a Risk Factor For Type 2 Diabetes. A 20 Year Population Based Prospective Study. Eur J Endocrinol 140 [suppl 1]:22 (abstract), 1999. 47: 15 19-1 524, 1998. 149 84. Amlov, J., Lind, L., Zethelius, B., Andrkn, B . , Hales, N. & Lithell, H. Indices related to insulin resistance are predictors of left ventricular systolic dysfunction in a male popu- lation after twenty years of follow-up. Eur J Endocrinol 142 [suppl 1]:17 (abstract), 2000. Address f o r correspondence: Hans Lithell, MD, PhD Department of Public Health and Caring Sciences/Geriatrics Uppsala University Box 609 SE-75 1 25 Uppsala Sweden 150