Decreased non-esterified fatty acid suppression and features of the insulin resistance syndrome occur in a sub-group of individuals with normal glucose tolerance

Diabetologia, Nov 1995

Summary To investigate causes of increased triglyceride concentrations in subjects with normal glucose tolerance (determined by oral glucose tolerance testing using World Health Organization criteria) 883 healthy subjects (389 men and 494 women) between 40 and 65 years of age were studied. Subjects were divided by gender into four groups according to 120-min glucose concentrations. Individuals in the highest quartile of glucose concentration had the highest mean triglyceride concentrations (p<0.0001) and highest mean non-esterified fatty acid (NEFA) concentrations (p<0.0001). There was also a clustering of cardiovascular risk factors normally associated with the insulin resistance syndrome in subjects in this group. Regression analysis showed that the most important determinants of triglyceride levels were smoking (men p=0.001, women p=0.005), waist:hip ratio (men p=0.01, women p<0.001) and NEFA suppression (men p=0.02, women p=0.005). NEFAs suppressed 16.7% in women compared to 2.4% in men during the first 30 min of the oral glucose tolerance test (p<0.001). These results show that a clustering of cardiovascular risk factors associated with decreased NEFA suppression occurs in a sub-group of subjects with normal glucose tolerance and that the pattern of NEFA suppression differs between men and women.

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Decreased non-esterified fatty acid suppression and features of the insulin resistance syndrome occur in a sub-group of individuals with normal glucose tolerance

Diabetologia Decreased non-esterified fatty acid suppression and features of the insulin resistance syndrome occur in a sub-group of individuals with normal glucose tolerance C.D. Byrne 0 N.J. Wareham 0 N.E. Day 0 R. McLeish 0 D. R. R. Williams 0 C.N. Hales ~ 0 0 1University Department of Clinical Biochemistry,Addenbrookes Hospital , Cambridge , UK 2Department of Community Medicine, Institute of Public Health, University of Cambridge , Cambridge , UK Summary To investigate causes of increased triglyceride concentrations in subjects with normal glucose tolerance (determined by oral glucose tolerance testing using World Health Organization criteria) 883 healthy subjects (389 m e n and 494 women) between 40 and 65 years of age were studied. Subjects were divided by gender into four groups according to 120rain glucose concentrations. Individuals in the highest quartile of glucose concentration had the highest m e a n triglyceride concentrations (p < 0.0001) and highest mean non-esterified fatty acid (NEFA) concentrations (p < 0.0001). There was also a clustering of cardiovascular risk factors normally associated with the insulin resistance syndrome in subjects in this group. Regression analysis showed that the most important determinants of triglyceride levels were smoking (men p = 0.001, women p = 0.005), waist:hip ratio (men p = 0.01, w o m e n p < 0.001) and NEFA suppression (men p = 0.02, women p = 0.005). NEFAs suppressed 16.7 % in women compared to 2.4 % in m e n during the first 30 min of the oral glucose tolerance test (p < 0.001). These results show that a clustering of cardiovascular risk factors associated with decreased N E F A suppression occurs in a sub-group of subjects with normal glucose tolerance and that the pattern of N E F A suppression differs between m e n and women. [Diabetologia (1995) 38: 1358-1366] Hypertriglyceridaemia; non-esterified fatty acid; ischaemic heart disease; smoking - 9 Springer-Verlag1995 Increased plasma triglyceride and decreased plasma high density lipoprotein (HDL) concentrations are cardinal features of the dyslipidaemia associated with the insulin resistance syndrome (IRS) [ 1 ]. Abnormalities of these Iipids may have important implications for the pathogenesis of ischaemic heart disease (IHD) both in subjects with N I D D M [ 2, 3 ] and in subjects with normal glucose tolerance [4]. Although it has been shown that hypertriglyceridaemia often occurs in association with hyperinsulinaemia and several of the other features of the IRS [ 5 ], little is known about the mechanisms causing abnormal triglyceride concentrations in subjects with normal glucose tolerance in the absence of inherited disorders of lipoprotein metabolism. Most of the triglyceride in the circulation in the fasting state originates from the very low density lipoprotein (VLDL) particles and both insulin and non-esterified fatty acids (NEFAs) have been shown by in vitro studies to markedly affect hepatic triglyceride secretion [ 6-9 ]. Whether hyperinsulinaemia per se causes hypertriglyceridaemia is still uncertain although, by contrast, NEFAs have been shown to be an important substrate and stimulus to hepatic triglyceride secretion. Inadequate suppression of NEFAs is associated with increased triglyceride concentrations and in vitro data support the notion that, in vivo, increased NEFAs may result in increased fasting triglyceride concentrations [ 10, 11 ], Smoking is also associated with increased triglyceride concentrations and the immediate effect of smoking is to increase lipolysis [ 12 ] although w h e t h e r smoking alters N E F A suppression during an oral glucose tolerance test (OGTT) is uncertain. The aims of this study were to: 1) determine the effects of NE FA s on triglyceride concentrations in the presence of insulin resistance and/or insulin deficiency and: 2) determine if increased triglyceride concentrations associated with smoking were attributable to increased NEFAs, in subjects who fulfilled World H e a l t h Organization criteria for normal glucose tolerance by OGTT. Subjects and methods Study design and subjects. The Isle of Ely Diabetes Project is a prospective population-based study of the aetiology and pathogenesis of N I D D M . Subjects for the study were recruited from a general practice register in Ely, Cambridgeshire, UK. The methods used in this study have previously b e e n described in detail [ 13 ]. Briefly, a letter of invitation was sent to a r a n d o m selection of patients not previously known to have diabetes, between the ages of 40 and 65 years at time of recruitment. Of the 1571 subjects invited, 1156 (74 %) participated and subjects taking oral contraceptives or h o r m o n e replacement therapy were not excluded from the study. A f t e r a 14-h fast subjects attended a clinical examination which included a dietary and medical questionnaire, anthropometric measurements and an O G T r . The analysis in this r e p o r t was restricted to the 883 subjects with normal glucose tolerance and subjects with either newly-diagnosed N I D D M or impaired glucose tolerance were excluded from the analysis. Smoking and alcohol consumption were assessed using H e a l t h and Lifestyle Survey study criteria [ 14 ]. Physical measurements. A n t h r o p o m e t r i c measurements (height, weight, waist and hip circumferences) were taken without shoes and in light clothing. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. Waist circumference was measured at the mid-point between the inferior b o r d e r of the costal margin and the anterior superior iliac spine and hip circumference at the level of the greater trochanters. Oral glucose tolerance test and biochemical analyses. The O G T T consisted of a 75-g oral glucose challenge with collection of venous blood samples at baseline, 30 rain and 120 rain. Samples of sera and plasma were immediately separated, kept on ice and stored a t - 7 0 ~ within 4 h. Plasma glucose was measured by a hexokinase m e t h o d [ 15 ] and triglyceride measured using the R A 1000 (Bayer Diagnostics, Basingstoke, U K ) , with a standard enzymatic method. Plasma insulin level was determined by two-site immunometric assays with either 125I or alkaline phosphatase labels [ 16, 17 ]. Insulin concentrations were measured in baseline, 30-rain and 120-rain samples. The 30-rain insulin increment was included in the analysis and was calculated by dividing the difference between 30-rain insulin and fasting insulin concentrations by the 30-min glucose concentration [10]. W H O criteria [ 18 ] were used to identify subjects with diabetes (fasting plasma glucose _>7.8 mmol/l or 2-h glucose _>11.1 mmol/1), impaired glucose tolerance (2-h glucose between 7.8-11.1 mmol/1) and normal glucose tolerance (2-h glucose < 7.8 mmol/1). Plasma N E F A measurements were d e t e r m i n e d enzymatically based on the activity of acyl-CoA synthetase (Boehringer Mannheim, Lewes, Sussex, UK). The resultant acyl-Co A is oxidised to yield hydrogen peroxide which is measured color metrically [ 19 ]. The assay had a between-assay coefficient of variation of 10 % at 0.40 mmol/1 and 6 % between 1.2 and 2.3 retool/1. N E F A concentrations were measured in baseline, 30-min and 120-min samples. N E F A area was also included in the analysis as a measure of both fasting N E F A concentration and suppression of NEFAs during the OGTT. N E F A area was calculated as the area under the trapezium described by the N E F A measurements at time 0, 30 and 120 min. The units of this measure are h . mmo1-1 1-I. The percentage of N E F A suppression was calculated over the first 30 rain of the OGTT. This value was obtained by subtracting the 30-rain N E F A concentration from the fasting N E F A concentration and dividing the resulting value by the fasting N E F A concentration. The number obtained was then expressed as a percentage. Statistical analysis The means of the baseline variables are presented in Tables 1 and 2, stratified by gender and quartile of plasma glucose at 120 rain. Arithmetic means ( + SD) are presented where the underlying variable is normally distributed and geometric mean (and 95 % confidence interval) where the distribution of a variable is skewed (Table 3). The Z test was used to compare means between genders within each quartile of plasma glucose concentration. Percentages of N E F A suppression for men and women were compared using a Mann-Whitney Utest. Spearman rank correlation coefficients (Table 4) were derived to show the relationship between variables stratified by category for non-smokers, ex-smokers and current smokers. Finally multiple regression models were derived for men and women separately to explain variation in triglyceride concentrations, variation in 120-min glucose concentrations and variation in H D L cholesterol concentrations. The following variables were considered for the triglyceride models; age, BMI, waist-hip ratio (WHR), smoking, alcohol intake, 120-min glucose level, N E F A area, fasting insulin and insulin increment. The same variables (excluding 120-min glucose level) were considered for the 120-rain plasma glucose model and the same variables including triglyceride were considered for the H D L cholesterol models. For each variable in the final model the standardised/3 coefficient and its significance are given (Tables 4-6). The overall adjusted R2 for each model allows comparison between models that include different numbers of explanatory variables. In these analyses the smoking category was coded f o r by a pair of binary indicator variables labelled sl and s2 [ 20 ]. A n indicator variable allows comparison between categories in a multiple regression analysis without imposing an artificial order on these groups. In this instance two variables sl and s2 are needed to describe the three categories: non-smokers (0,0), ex-smokers (1,0) and smokers (0,1). Results A t o t a l o f 3 8 9 m e n a n d 4 9 4 w o m e n w e r e i d e n t i f i e d w i t h n o r m a l g l u c o s e t o l e r a n c e as d e f i n e d b y W H O c r i t e r i a . I n o r d e r t o d e t e r m i n e if a l t e r e d N E F A s u p p r e s s i o n a n d f e a t u r e s n o r m a l l y a s s o c i a t e d w i t h t h e I R S w e r e a f f e c t e d b y p l a s m a g l u c o s e c o n c e n t r a t i o n s , s u b j e c t s w e r e d i v i d e d i n t o f o u r g r o u p s o n t h e b a s i s C.D. B y r n e et al.: H y p e r t r i g l y c e r i d a e m i a in subjects w i t h n o r m a l glucose t o l e r a n c e T a b l e 3, A n t h r o p o m e t r i c m e a s u r e s , b i o c h e m i c a l results a n d s m o k i n g s t a t u s Non-smokers (a) Ex-smokers (b) C u r r e n t smokers (c) C o m p a r i s o n of means ab ac bc 0.025 0.031 0.022 NS NS NS NS NS NS NS NS NS R e s u l t s a r e S p e a r m a n r a n k c o r r e l a t i o n coefficients (significance); a p < 0.05; b p < 0.0l; Cp < 0.001; P G 1 2 0 , P l a s m a glucose at 120 m i n (mmol/1); I n s u l i n 0', f a s t i n g insulin c o n c e n t r a t i o n (pmol/1); H D L chol, H D L c h o l e s t e r o l (mmol/1) C,D. Byrne et al.: Hypertriglyceridaemia in subjects with normal glucose tolerance sl and s2 are dummy variables grouping subjects into non-smokers, ex-smokers and current smokers sl and s2 are dummy variables grouping subjects into non-smokers, ex-smokers and current smokers of their 120-min p l a s m a glucose c o n c e n t r a t i o n s (Tables 1 and 2). In these analyses p-values were calculated to d e t e r m i n e if significant trends existed across the four groups. Tables 1 a n d 2 include c o m p l e t e data o n 876 of 883 subjects with n o r m a l glucose tolerance. N E F A area (a m a r k e r of N E F A suppression) increased with increasing p l a s m a glucose in b o t h sexes and fasting N E F A c o n c e n t r a t i o n s were increased in w o m e n c o m p a r e d with m e n (Fig. 1). In o r d e r to determ i n e if N E F A suppression was different b e t w e e n the sexes the p e r c e n t a g e of N E F A suppression was calculated for m e n and w o m e n during the first 30 rain of the O G T T . In m e n the m e d i a n p e r c e n t a g e suppression during the first 30 m i n was 2.4 9/0 whereas in wom e n the m e d i a n p e r c e n t a g e suppression was 16.7 % (p < 0.001). To d e t e r m i n e if t h e r e were differences b e t w e e n triglyceride c o n c e n t r a t i o n s in individuals w h o were c u r r e n t smokers, ex-smokers and non-smokers, triglyceride c o n c e n t r a t i o n s were c o m p a r e d in the t h r e e groups (Table 3). S p e a r m a n r a n k correlation coefficients were calculated for triglyceride, N E F A area, 120-rain plasma glucose and fasting insulin for b o t h m e n and w o m e n w h o were smokers, ex-smokers and n o n - s m o k e r s (Table 4). In o r d e r to investigate associations b e t w e e n possible explanatory variables and triglyceride concentrations multiple regression analyses were u n d e r t a k e n . In the first regression m o d e l with p l a s m a triglyceride as the d e p e n d e n t variable, N E F A area, W H R and cigarette s m o k i n g were i n d e p e n d e n t l y associated with triglyceride concentrations in b o t h sexes (Table 5). As it was likely that N E F A suppression was affected by insulin concentrations this regression m o d e l was r e p e a t e d with fasting insulin (as a m e a s u r e of insulin resistance) and 30-min insulin i n c r e m e n t (as a m e a s u r e of insulin secretion) a d d e d to the model. In this m o d e l N E F A area was still i n d e p e n d e n t l y associated with triglyceride levels (for m e n fl coefficient 0.15, p < 0.01; and for w o m e n fl coefficient 0.08, p < 0.05). Fasting insulin was i n d e p e n d e n t l y assocP ated with triglyceride levels in b o t h sexes (for m e n fl coefficient 0.18, p < 001; and for w o m e n fl coefficient 0.3, p < 0.001) whereas no association with 30-min insulin i n c r e m e n t was f o u n d in either sex. A d d i n g b o t h o ; 3'o do 9'0 Time (min) Fig.lA, B. Plasma NEFA concentrations at 0, 30 and 120 rain after administration of 75-g oral glucose in males (A) and females (B), stratified by 120-rainpost-load plasma glucose concentration (PG)I -[~-, PG < 5; -o-, PG = 5-5.69; - B , PG = 5.7 - 6.49; -t~, PG _>6.5 mmol/1 fasting insulin and insulin increment to this model did not adjust the R 2 value for either sex. To determine if plasma glucose affected the independent association between N E F A area (as a marker of N E F A suppression) and triglyceride concentrations, 120-min plasma glucose was introduced into the regression model with plasma triglyceride as the d e p e n d e n t variable. In this analysis fasting insulin and 30-min insulin increment were excluded from the model (Table 6). The results show that the strength of the association b e t w e e n triglyceride and N E F A area was reduced and that glucose concentration was independently associated with triglyceride concentration in both sexes. Therefore, to further investigate the association between 120-min plasma glucose and N E F A area, regression analysis was undertaken with 120-min glucose as the dependent variable (Table 7). The same analysis was repeated excluding N E F A area from the model to determine if the strength of the associations between insulin parameters (as markers of insulin resistance and insulin deficiency) and 120-min plasma glucose were altered. In m e n the adjusted R 2 changed from 0.13 to 0.09 whereas in w o m e n the adjusted R 2 changed from 0.23 to 0.16 indicating that N E F A suppression explained some of the variation in 120-rain glucose independently of insulin concentrations. H D L cholesterol was inversely associated with triglyceride levels in non-smokers, ex-smokers and current smokers (Table 4). To examine the association between H D L cholesterol and triglyceride, regression analysis was u n d e r t a k e n with H D L cholesterol as the dependent variable. In m e n the adjusted R 2 w a s 0.24 and triglyceride (inversely) and alcohol were the only factors independently associated with H D L (~ coefficients -0.37 and 0.24, respectively, both p < 0.0001). In w o m e n the adjusted R 2 w a s 0.22 and, triglyceride (inversely), W H R (inversely) and age were independently associated with H D L (/3 coefficients -0.35, -0.19 and 0.23, all p < 0.0001). A n analysis of anthropometric measures and biochemical results u n d e r t a k e n in non-smokers, exsmokers and current smokers showed that there were differences in metabolic risk factors between the groups (Table 3). To determine the effect of smoking on triglyceride concentrations, regression analysis was u n d e r t a k e n with triglyceride as the dependent variable in two different models (Tables 5 and 6). As differences in anthropometric and biochemical measures existed between groups according to smoking status, the d u m m y variables sl and s2 were introduced into these m o d e l s to represent subjects grouped into non-smokers, ex-smokers and current smokers. The results show that smoking was associated with triglyceride levels independently of alcohol consumption, age, p l a s m a glucose, BMI, W H R and N E F A area. To determine whether the effect of smoking on triglyceride concentrations was mediated through changes in N E F A suppression regression analysis was u n d e r t a k e n with N E F A area as the dependent variable and the same d u m m y variables sl and s2 representing smoking category. This analysis showed that there was no independent association between smoking and N E F A area in both sexes. Fasting insulin and 30-min insulin increment were also introduced into the regression model with triglyceride as the dependent variable (i. e., Table 5) to determine whether insulin secretion or insulin resistance interacted with smoking to affect plasma triglyceride concentrations. In this analysis smoking was still independently associated with triglyceride levels in both sexes (for m e n /3 coefficient 0.23, p < 001; and for w o m e n ~ coefficient 0.14, p = 0.001), indicating that addition of insulin parameters to the model did not explain any m o r e of the variation in plasma triglyceride as the model adjusted R 2 values remained unchanged. sl and s2 are dummy variables grouping subjects into non-smokers, ex-smokers and current smokers Discussion The results of this study show that there is a clustering of risk factors normally associated with IRS in subjects within the highest quartile of 120-min glucose concentrations. Therefore, a sub-group of individuals who satisfy the W H O criteria for normal glucose tolerance may be at increased risk from I H D because they have many of the features normally associated with IRS. This supports the concept of treating glucose tolerance as a continuous variable and suggests that placing an individual in a group entitled "normal glucose tolerance" may disguise the fact that an individual is still at risk from IHD. These results also show that there were marked differences between subjects grouped according to quartites of 120-rain glucose in N E F A suppression (as measured by N E F A area) in both sexes. Individuals of both sexes in the top quartile had markedly increased NEFAs compared to individuals in the b o t t o m quartile and failure to suppress NEFAs during O G T T and an increased W H R were both associated with raised plasma triglyceride concentrations. At 120 min NEFAs were completely suppressed and although in our study the 30-min measurement helped to determine differences in N E F A suppression between the groups it is likely that a m o r e accurate determination of N E F A suppression could be achieved by additional measurements around the 30-min time point. The percentage of N E F A suppression during the first 30 min of the O G T T showed that in w o m e n N E F A suppression was more marked than in m e n and greater N E F A suppression may contribute to lower triglyceride concentrations in women. We have shown previously that N E F A suppression was reduced during the first 30 min of an O G T T in wom e n with newly-diagnosed N I D D M [ 10 ] compared to w o m e n with normal glucose tolerance and it is possible that (in women) both diabetes and a male pattern of fat distribution reduce rapid suppression of NEFAs, resulting in increased plasma triglyceride concentrations. In order to study associations between 120-min glucose, insulin resistance, relative insulin deficiency and N E F A area, regression analysis was undertaken with 120-rain plasma glucose as the dependent variable. Fasting insulin was included in the model as a surrogate for insulin resistance [ 21 ], the 30-min insulin increment was included as a surrogate for insulin deficiency [ 10, 22 ] and N E F A area was included as a surrogate for N E F A suppression [10]. The results showed that N E F A area was strongly associated with 120-rain glucose in the regression analysis. As expected both fasting insulin and 30-min insulin increment were independently associated with 120min plasma glucose. Exclusion of N E F A area from the analysis weakened the model and explained less of the variation in glucose concentrations indicating that insulin concentrations alone did not explain all of the variation in 120-min glucose. These results support the notion that increased N E F A concentrations may worsen insulin resistance through the Randle cycle [ 23 ]. Recently it has been shown that NEFAs decreased glycogen synthesis and carbohydrate oxidation and reduced the insulin-mediated decrease in hepatic glucose output [ 24 ]. Increased NEFAs may also be intimately linked t o increased triglyceride levels since NEFAs increase hepatic trigtyceride synthesis and V L D L production [ 7, 8 ] and we have also shown that NEFAs may increase hepatic triglyceride secretion by reduction of the inhibitory effect of insulin on hepatic triglyceride secretion [8]. This latter effect of NEFAs has recently been shown to occur in vivo [ 25 ]. In individuals with normal glucose tolerance V L D L production increased in response to an increase in plasma NEFAs. Infusion of insulin-reduced V L D L secretion and the insulin-induced decrease in V L D L production was also diminished by concomitant administration of fatty acid. Thus, the negative effect of NEFAs on insulin action appears to represent another aspect of NEFAs ability to act as insulin antagonists as originally proposed with the concept of the Randle cycle. W h e n fasting insulin level (a m a r k e r of insulin resistance) was introduced into the regression model with triglyceride as the d e p e n d e n t variable the independent association of N E F A area with triglyceride concentration remained, suggesting that insulin resistance to glucose disposal alone does not determine N E F A concentrations. Increased plasma N E F A concentrations may also serve as a m a r k e r for worsening glucose tolerance, although failure of insulin to adequately suppress hormone-sensitive lipase probably does not explain all of the association between glucose and N E F A area, as catecholamines also affect lipolysis. It has been shown that in subjects with increased W H R , catecholamine-induced lipolysis was reduced in subcutaneous adipose tissue biopsies from male subjects with IRS [ 26 ]. A g e was a major determinant of triglyceride levels in females only. Since age was an unimportant explanatory variable in m e n a possible effect of the menopause is suggested. The m e a n age of w o m e n in the lowest quartile of triglyceride levels was 49.4 years and in the highest quartile was 55.6 years. The menopausal status of our subjects was not known but it is likely that some of the individuals from the lowest quartile were pre-menopausal and those from the highest quartile are post-menopausal as the average age of m e n o p a u s e in the U K is 50 years [ 27 ]. Although our data are cross-sectional they suggest that the menopause m a y affect triglyceride levels independently of age, as age was not an ind e p e n d e n t determinant of triglyceride concentrations in men. This observation accords with recent work showing that triglyceride concentrations increased independently of age with the menopause [ 28 ]. The mechanism causing increased triglyceride concentrations is unknown and whether menoPause or age directly affects N E F A suppression is uncertain. Recently it has been shown that menopause increased fasting NEFAs in Asian w o m e n but no effect of m e n o p a u s e on fasting NEFAs was shown in Caucasian w o m e n [ 11 ]. Smoking was also associated with increased plasm a triglyceride concentrations. Smoking was associated with a higher W H R in both sexes and a higher BMI in w o m e n but not in men. Recently it has been shown that an immediate effect of smoking is to increase plasma N E F A concentrations by increasing N E F A entry into the circulation and by increasing hepatic re-esterification [ 12 ]. Our results showed that in the regression analysis after adjustment, smoking was not independently associated with any alteration of N E F A suppression. Subjects in our study were asked not to smoke after 22.00 hours during the evening preceding the day of the test and were not allowed to smoke until the test was completed. Consequently, the results of our study are not directly comparable to those of Hellerstein et al. [ 12 ], as it is likely that any independent effect of smoking on triglyceride in our study would be attributable to a longerterm effect. Since the regression analysis with triglyceride as the d e p e n d e n t variable showed that the effect of smoking on triglyceride in current smokers was different from that in ex-smokers, it is likely that the effect of smoking on triglyceride concentration is a m e d i u m - t e r m effect and does disappear with time. Our results showed that N E F A area was lower in current smokers and ex-smokers compared to control subjects but it is likely that this is attributable to lower plasma glucose concentrations in smokers and exsmokers as glucose levels were strongly associated with N E F A area. A t present we are unable to explain the lower glucose concentrations in smokers and ex-smokers compared to non-smokers. Despite the lower plasma glucose concentrations and lower N E F A areas in smokers compared to non-smokers, triglyceride levels were higher in this group compared to control subjects. Regression analysis showed that the effect of smoking on triglycerides was independent of alcohol consumption, age, 120min plasma glucose, BMI, W H R and N E F A area and therefore suggests that the effect of smoking is mediated by an alternative unknown mechanism. In conclusion, 53 (6 %) of the subjects (41 m e n and 12 women) participating in this study had H D L cholesterol concentrations of below 1.02 mmol/1 and were in the highest quartile of triglyceride concentration (i. e., _>1.8 mmol/1 in m e n and >_1.4 mmol/1 in women). The Framingham study has shown that similar, relatively mild perturbations of both lipids were associated with a m a r k e d increase in I H D risk, that according to Castelli [ 4 ], places these individuals on the "fast track to atherosclerotic disease". Our data show that in these "at-risk" individuals plasma glucose was increased compared to the rest of the sample, there was a m a r k e d decrease in NIEFA suppression and these two associated abnormalities m a y have significant implications for triglyceride concentrations in such subjects. According to the 1991 census data there are approximately 14.6 million individuals between the ages of 40 and 65 years in the U K [ 29 ]; 6 % of 14.6 million is 880,000 at-risk individuals. Attempts to identify these individuals m a y be beneficial as simple measures to reduce abdominal girth, increase physical activity and stop smoking m a y significantly reduce their risk of IHD. Acknowledgements. This study was supported by grants from the BDA and MRC, Bayer Diagnostics, Corda and Lilley Research Laboratories. C.D.B. is an MRC Clinician Scientist. The technical assistance of R. Beck, L. Cox, T. Elsey, N.D. Martensz, B. Mission, M. Sheldon and A. E M. Tullock is gratefully acknowledged. We would like to thank D. Brown, E Clark, B. Cox, J.M. Lipscombe, M. Quinn, S. Farmer, L. Koncewicz, C. Palmer, H. Shanassy and T. Wang for their assistance in the field work and data entry. Finally we are grateful to all the volunteers who participated in the study and to Dr. J. 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C. D. Byrne, N. J. Wareham, N. E. Day, R. McLeish, D. R. R. Williams, C. N. Hales. Decreased non-esterified fatty acid suppression and features of the insulin resistance syndrome occur in a sub-group of individuals with normal glucose tolerance, Diabetologia, 1995, 1358-1366, DOI: 10.1007/BF00401770