Circulating Fatty Acids, Non-High Density Lipoprotein Cholesterol, and Insulin-Infused Fat Oxidation Acutely Influence Whole Body Insulin Sensitivity in Nondiabetic Men

The Journal of Clinical Endocrinology & Metabolism, Feb 2005

Circulating lipids and tissue lipid depots predict insulin sensitivity. Associations between fat oxidation and insulin sensitivity are variable. We examined whether circulating lipids and fat oxidation independently influence insulin sensitivity. We also examined interrelationships among circulating lipids, fat oxidation, and tissue lipid depots. Fifty-nine nondiabetic males (age, 45.4 ± 2 yr; body mass index, 29.1 ± 0.5 kg/m2) had fasting circulating nonesterified fatty acids (NEFAs) and lipids measured, euglycemic-hyperinsulinemic clamp for whole body insulin sensitivity [glucose infusion rate (GIR)], substrate oxidation, body composition (determined by dual energy x-ray absorptiometry), and skeletal muscle triglyceride (SMT) measurements. GIR inversely correlated with fasting NEFAs (r = −0.47; P = 0.0002), insulin-infused NEFAs (n = 38; r = −0.62; P < 0.0001), low-density lipoprotein cholesterol (r = −0.50; P < 0.0001), non-high-density lipoprotein cholesterol (r = −0.52; P < 0.0001), basal fat oxidation (r = −0.32; P = 0.03), insulin-infused fat oxidation (r = −0.40; P = 0.02), SMT (r = −0.28; P < 0.05), and central fat (percentage; r = −0.59; P < 0.0001). NEFA levels correlated with central fat, but not with total body fat or SMT. Multiple regression analysis showed non-high-density lipoprotein cholesterol, fasting NEFAs, insulin-infused fat oxidation, and central fat to independently predict GIR, accounting for approximately 60% of the variance. Circulating fatty acids, although closely correlated with central fat, independently predict insulin sensitivity. Insulin-infused fat oxidation independently predicts insulin sensitivity across a wide range of adiposity. Therefore, lipolytic regulation as well as amount of central fat are important in modulating insulin sensitivity.

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Circulating Fatty Acids, Non-High Density Lipoprotein Cholesterol, and Insulin-Infused Fat Oxidation Acutely Influence Whole Body Insulin Sensitivity in Nondiabetic Men

The Journal of Clinical Endocrinology & Metabolism Circulating Fatty Acids, Non-High Density Lipoprotein Cholesterol, and Insulin-Infused Fat Oxidation Acutely Influence Whole Body Insulin Sensitivity in Nondiabetic Men A. M. Poynten 0 1 S. K. Gan 0 1 A. D. Kriketos 0 1 L. V. Campbell 0 1 D. J. Chisholm 0 1 0 First Published Online 1 Diabetes and Obesity, Garvan Institute of Medical Research , Sydney 2010 , Australia Circulating lipids and tissue lipid depots predict insulin sensitivity. Associations between fat oxidation and insulin sensitivity are variable. We examined whether circulating lipids and fat oxidation independently influence insulin sensitivity. We also examined interrelationships among circulating lipids, fat oxidation, and tissue lipid depots. Fifty-nine nondiabetic males (age, 45.4 2 yr; body mass index, 29.1 0.5 kg/m2) had fasting circulating nonesterified fatty acids (NEFAs) and lipids measured, euglycemic-hyperinsulinemic clamp for whole body insulin sensitivity [glucose infusion rate (GIR)], substrate oxidation, body composition (determined by dual energy x-ray absorptiometry), and skeletal muscle triglyceride (SMT) measurements. GIR inversely correlated with fasting NEFAs (r 0.47; P 0.0002), insulin-infused NEFAs (n 38; r 0.62; P < 0.0001), low-density lipoprotein cholesterol (r 0.50; P < 0.0001), non-high-density lipoprotein choles- - T HE DEVELOPMENT OF insulin resistance is closely associated with obesity and is a precursor to the development of type 2 diabetes. The mechanisms by which obesity leads to insulin resistance are still not entirely understood. There is evidence that lipid tissue depots influence insulin sensitivity ( 1–3 ). The role of circulating lipids and fat oxidation as acute modulators of insulin resistance is of renewed interest. Peroxisomal proliferator-activated receptor- and - agonists, which improve insulin resistance in humans, alter both circulating fatty acids levels and skeletal muscle oxidation of fatty acids ( 4, 5 ). It has been suggested that increased visceral fat may lead to an associated rise in portal vein plasma fatty acid concentrations. It was reported some years ago that increased delivery of fatty acids to the liver might lead to elevated insulin levels by decreasing insulin clearance ( 6 ). Recent studies using parenteral elevation of fatty acids have confirmed increased endogenous glucose production and decreased clearance of insulin ( 7 ) [without increased insulin secretion ( 8 )], resulting in relative systemic hyperinsulinJCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community. terol (r 0.52; P < 0.0001), basal fat oxidation (r 0.32; P 0.03), insulin-infused fat oxidation (r 0.40; P 0.02), SMT (r 0.28; P < 0.05), and central fat (percentage; r 0.59; P < 0.0001). NEFA levels correlated with central fat, but not with total body fat or SMT. Multiple regression analysis showed non-high-density lipoprotein cholesterol, fasting NEFAs, insulin-infused fat oxidation, and central fat to independently predict GIR, accounting for approximately 60% of the variance. Circulating fatty acids, although closely correlated with central fat, independently predict insulin sensitivity. Insulininfused fat oxidation independently predicts insulin sensitivity across a wide range of adiposity. Therefore, lipolytic regulation as well as amount of central fat are important in modulating insulin sensitivity. (J Clin Endocrinol Metab 90: 1035–1040, 2005) emia. Elevated systemic circulating fatty acid levels have been associated with increased risk of developing type 2 diabetes ( 9 ). Experimental elevation of nonesterified fatty acids (NEFAs) in animals and humans leads to accumulation of intramyocellular lipid and skeletal muscle insulin resistance ( 10 ). Elevation of circulating fatty acids has also been shown to influence insulin signaling pathways in skeletal muscle ( 11 ). In limb balance studies, obese insulin-resistant humans have been shown to have reduced skeletal muscle fat oxidation in the basal state ( 12 ). This may be partly responsible for the development of increased intramyocellular lipid with its adverse effect on insulin sensitivity ( 13 ). Obese insulinresistant humans also have defective suppression of fat oxidation under insulin-infused conditions ( 14 ), which may expose insulin-sensitive tissues to high NEFA levels in the postprandial state. The aim of the current study was to determine whether circulating lipids, fatty acids, and fat oxidation predict whole body insulin sensitivity independently of abdominal fat, total body fat, and muscle lipid depots. A second aim was to examine the interrelationships among circulating lipids, fat oxidation, and tissue lipid depots. Subjects and Methods Caucasian male volunteers (n 59) were recruited from advertisements in the local press. Male subjects were chosen to avoid the confounding effects of menopausal status and oral contraceptive use on insulin sensitivity ( 15 ). Volunteers were excluded if there was a history of diabetes mellitus or cardiovascular, renal, or other clinically significant disease. The St. Vincent’s Hospital research ethics committee approved the protocol, and all subjects gave written informed consent. The study protocol was conducted in accordance with the Helsinki Declaration. Before entry into the study, fasting plasma glucose was measured. Volunteers with plasma glucose greater than 5.6 mmol/liter had a 75-g glucose tolerance test. Glucose tolerance was defined according to the World Health Organization criteria ( 16 ). Only men who were not diabetic took part in the study. Subjects were asked to consume their usual diet and to refrain from alcohol and vigorous exercise for at least 3 d before the study. Metabolic assessments were performed on a single day. After an overnight fast, anthropometric measurements were performed, followed by skeletal muscle biopsy of vastus lateralis and euglycemic-hyperinsulinemic clamp. Indirect calorimetry was performed in the resting state, before the clamp, and in the final 30 min of the clamp. After a light lunch, subjects had whole body dual energy x-ray absorptiometry (DEXA) performed to assess body composition. Skeletal muscle biopsy All subjects underwent skeletal muscle biopsy. Under local anesthesia, a 6-mm diameter University College Hospital muscle biopsy needle was inserted into the vastus lateralis muscle (15 cm above the patella), yielding samples of approximately 200 mg. Biopsied muscle tissue was immediately immersed in liquid nitrogen and stored at 80 C. Euglycemic-hyperinsulinemic clamp and indirect calorimetry Height was measured with a stadiometer, and weight was determined with calibrated scales. Body mass index (BMI) was calculated (kilograms per meter squared). All subjects underwent a euglycemichyperinsulinemic clamp for assessment of insulin sensitivity. Clamp studies were started at 0830 h after skeletal muscle biopsy. An iv catheter was inserted under local anesthesia into an antecubital vein for infusion of human insulin (Actrapid, Novo Nordisk, Copenhagen, Denmark) and glucose. A second iv catheter was inserted into a warmed contralateral forearm vein and was used for arterialized blood sampling. Fasting blood samples were obtained for measurement of lipid [total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride (TG), NEFA, and total apolipoprotein B (apoB)], glucose, and insulin levels. Insulin was infused at 50 mU/m2 surface area min for 150 min, producing insulin levels in the high physiological range ( 90 mU/liter), which have been shown to suppress hepatic glucose output in healthy humans ( 17 ). Clamp studies employing a slightly lower dose of insulin (45 mU/m2 min) and using a glucose tracer have found complete suppression of endogenous glucose production in all subjects except those with a fasting glucose level greater than 9.7 mmol/liter ( 18 ). None of our subjects had a fasting glucose level greater than 7 mmol/liter, so endogenous glucose production was presumed to be suppressed. Blood samples were taken every 10 min for assessment of glucose and insulin. At 150 min, plasma was stored at 80 C for measurement of NEFA levels (in 38 subjects). A variable glucose infusion rate was used to maintain blood glucose levels at 5 mmol/liter. The steady state glucose infusion rate (GIR) over the final 40 min of the clamp provided an index of whole body insulin sensitivity and was expressed as micromoles per minute per kilogram of fat-free mass (determined by DEXA). Indirect calorimetry (Deltatrac, Datex, Helsinki, Finland) was performed for 30 min before the clamp and at 120 –150 min during hyperinsulinemia as previously described ( 19 ). The coefficient of variation for measuring the respiratory quotient in our laboratory is approximately 4%. Resting metabolic rates and fat and carbohydrate oxidation rates (grams per minute) were calculated ( 20 ). Rates of protein oxidation were estimated based on the subject’s weight ( 21 ). Body composition Body composition was assessed using whole body DEXA (Lunar DPX, Lunar Radiation Corp., Madison, WI; software version 1.35y) ( 20 ). Total and regional tissue compositions, fat, muscle, and bone, were measured in grams and as a percentage of tissue. Fat-free mass was calculated as the sum of bone and muscle masses. A central abdominal region was defined as extending 9.8 cm in vertical dimension with the lower border at the superior iliac crest and with lateral dimensions at the lateral borders of the costal margins. Central fat measured by DEXA has a strong correlation with computer tomography measurement of visceral fat ( 22 ) and is strongly related to insulin sensitivity ( 23 ). Biochemical analysis Plasma glucose was measured by the oxidase method (NOVA 14, Nova Biomedical, Waltham, MA). Serum free insulin was assayed by RIA (Linco Research, Inc., St. Charles, MO). Blood samples for serum cholesterol, HDL cholesterol, and TG were collected in plain tubes (Becton Dickinson, Franklin Lakes, NJ), immediately spun in a refrigerated centrifuge (at 4 C), and stored at 20 C. Total cholesterol, HDL cholesterol, and TG concentrations were determined spectrophotometrically at 490 nm using enzymatic colorimetric kits (Roche, Basel, Switzerland). Low-density lipoprotein (LDL) cholesterol was estimated using the Friedewald formula ( 24 ). Non-HDL cholesterol was calculated as total cholesterol minus HDL cholesterol. Blood samples for total apoB were collected in BD SST 11 tubes (containing acrylic gel, silica clot surface activator) and were immediately spun in a refrigerated centrifuge (at 4 C) and stored at 20 C. Total serum apoB was measured by rate immunoturbometric method (Roche, Indianapolis, IN) and was quantified on a Hitachi 917 machine (Tokyo, Japan). Blood samples for NEFA measurement were collected in EDTA tubes, placed on ice, then spun in a refrigerated centrifuge (4 C). Plasma was separated, immediately frozen in liquid nitrogen, and stored at 80 C. NEFA levels were determined by enzymatic colorimetry (NEFA C kit, WAKO Pure Chemical Industries, Osaka, Japan). The inter- and intraassay coefficients of variation for NEFA were 5% and 5%, respectively, at 500 mol/liter. Skeletal muscle TG (SMT) content Approximately 50 mg skeletal muscle were freeze-dried under vacuum for 24 h. After freeze-drying, the muscle sample was viewed under a microscope ( 6.3) at room temperature for careful dissection and removal of all trace of adipose tissue, connective tissue, and blood contaminants. This yielded approximately 10 mg dry weight dissected skeletal muscle. The extraction of lipids from freeze-dried and carefully dissected muscle fibers and subsequent estimation of TG content are reproducible in our laboratory with a within-assay variability of approximately 8% ( 25 ). Statistical analyses Data were analyzed using StatView 5 (Abacus Concepts, Inc., Berkeley, CA). All values are given as the mean se. Associations between continuous variables were assessed using simple or multiple regression analyses as appropriate. P 0.05 was considered significant. Results Table 1 shows the clinical characteristics and clamp data of the 59 male participants. The mean BMI was in the overweight range. There were wide ranges of ages and adiposity (measured by DEXA). None of the participants had type 2 diabetes mellitus, but several had impaired fasting glucose. There was a wide range of insulin sensitivity, as measured by glucose infusion rate during stable hyperinsulinemia (mean steady state clamp insulin, 91.2 4.4 mU/liter). No subject had serum TG greater than 4 mmol/liter (which would render the Friedewald formula invalid). Whole body insulin sensitivity, as assessed by GIR, was negatively correlated with fasting NEFA, insulin-infused NEFA, fasting LDL cholesterol, and non-HDL cholesterol levels in the group (Table 2). These relationships with GIR were similar if the two subjects with lowest central fat (percentage) were excluded (for GIR and fasting NEFAs and GIR and LDL cholesterol: r 0.41; P 0.001; for GIR and clamp NEFAs: r 0.60; P 0.0001; for GIR and non-HDL cholesterol: r 0.43: P 0.001). There was a small, but significant, negative correlation between GIR and the difference between fasting and insulin-infused NEFA levels (r 0.03; P 0.04). The association between GIR and plasma TG was of borderline significance (Table 2). There was a significant negative correlation between whole body insulin sensitivity and basal fat oxidation and insulin-infused fat oxidation (Table 2). Central abdominal fat (percentage) and SMT were negatively associated with GIR (Table 2). There were no significant associations between GIR and fasting HDL cholesterol, TG:HDL ratio, total apoB, insulin, or glucose levels (data not shown). GIR was inversely related to age (r 0.50; P 0.01). Figure 1 shows the interrelationships between the lipid measurements by simple linear regression. Both fasting NEFA levels (Fig. 1A) and insulin-infused NEFA levels (r 0.40; P 0.01) were positively associated with central fat (percentage). LDL cholesterol and non-HDL cholesterol were also significantly associated with central fat (percentage; r 0.32; P 0.02 and r 0.35; P 0.01 respectively), but these relationships were not significant when the two subjects with lowest central fat (percentage) and low LDL cholesterol levels were excluded (r 0.13; P 0.33 and r 0.17; P 0.22 respectively). Serum TG and SMT were also associated with central fat (percentage; r 0.30; P 0.02 and r 0.27; P 0.04, respectively). Total fat (percentage) was not associated with fasting or insulin-infused NEFA levels, LDL cholesterol, non-HDL cholesterol, or basal or insulin-infused fat oxidation (data not shown). There was no significant relationship between central fat and basal fat oxidation (r 0.14; P 0.35). Figure 1B shows the significant relationship between fasting NEFAs and basal fat oxidation. There was a similar relationship between insulin-infused NEFAs and insulininfused fat oxidation (r 0.40; P 0.02). Fasting and insulininfused NEFAs were strongly correlated (r 0.62; P 0.0001). Fasting NEFAs had no association with fasting plasma insulin or glucose levels (r 0.04; P 0.76 and r 0.01; P 0.92, respectively) and were not significantly related to fasting TG levels (r 0.21; P 0.08). There was no relationship between clamp NEFA levels and fasting TG levels (r 0.12; P 0.46). LDL cholesterol and non-HDL cholesterol were not associated with fasting NEFAs, insulininfused NEFAs, or fat oxidation; there were no associations between SMT and fat oxidation and circulating lipids (data not shown). Age was associated with fasting NEFAs (r 0.46; P 0.001), insulin-infused NEFAs (r 0.37; P 0.03), basal fat oxidation (r 0.38; P 0.01), and SMT (r 0.45; P 0.001), but not with insulin-infused fat oxidation (r 0.11; P 0.15), LDL cholesterol (r 0.23; P 0.08), or non-HDL cholesterol (r 0.23; P 0.08). Age was also closely related to central fat (percentage; r 0.46; P 0.001), but not to total fat (percentage) or BMI (r 0.22; P 0.09 and r 0.08; P 0.53, respectively). The independent influences of the measured lipid parameters on insulin sensitivity were assessed by multiple regression analyses. In the whole group of 59 men, with glucose infusion rate as the dependent variable, central fat (percentage), non-HDL cholesterol, fasting NEFAs, and insulininfused fat oxidation were independently associated with whole body insulin sensitivity as measured by GIR, accounting for approximately 60% of the variance (Table 3). If LDL cholesterol was entered into the analysis instead of non-HDL cholesterol, LDL cholesterol was independently associated with GIR (with a standard coefficient of 0.33) and, with the other factors independently associated with GIR (central fat, fasting NEFAs, and insulin-infused fat oxidation), accounted for approximately 60% of the variance. Age, basal fat oxidation, total body fat (percentage), and SMT, which in simple correlation were correlated with GIR, were not significant when entered into the model. HDL cholesterol, total apoB, plasma TG, fasting plasma insulin, and fasting plasma glucose were not significantly associated with GIR in the multiple regression analysis in the whole group. Multiple regression analysis, with GIR as the dependent variable, was performed for the 38 subjects for whom insulininfused NEFAs were available. In that model, significant independent predictors of GIR were central fat (percentage; standard coefficient, 0.36; P 0.005), non-HDL cholesterol (standard coefficient, 0.34; P 0.01), and insulin-infused NEFAs (standard coefficient, 0.38; P 0.002), accounting for approximately 65% of the variance. Basal NEFAs, LDL cholesterol, and insulin-infused fat oxidation were not significant in this model. If LDL cholesterol was entered into the analysis instead of non-HDL cholesterol, it was independently associated with GIR (standard coefficient, 0.32; P 0.01). Discussion In our study of nondiabetic men, both fasting and insulininfused NEFAs were strongly associated with insulin resistance. Elevated fasting NEFA levels are a significant risk factor for the development of type 2 diabetes ( 9, 26 ). Therefore, elevated NEFA levels may increase the risk of later development of type 2 diabetes via a previous increase in insulin resistance. Fasting and insulin-infused NEFAs showed no relationship with fasting glucose or insulin, in accordance with other studies ( 9, 27, 28 ). Although in the study by Pankow et al. (9), there was no interaction with NEFAs or BMI, we found a strong relationship between fasting NEFAs and insulin-infused NEFAs and central fat measured by DEXA. This relationship may be due to the relative resistance of visceral fat cells to the anti-lipolytic effects of insulin ( 29 ) and higher rates of catecholaminestimulated lipolysis ( 30 ), which may lead to an increased release of fatty acids from visceral fat. However, in the whole group, basal NEFA levels were an independent factor for insulin sensitivity, suggesting an influence independent of central fat stores. All cross-sectional correlation studies have limitations in relation to causality. Thus, it is possible that increased NEFA levels reflect insulin resistance of adipocytes, with reduced uptake of fatty acids or increased lipolysis. However, the difference between basal and clamp NEFAs, attributable to the insulin infusion, was not an independent predictor for GIR. There are theoretical mechanisms for the contribution of circulating fatty acids to insulin resistance. In humans, parenteral and oral elevation of circulating fatty acids induces insulin resistance ( 10, 31 ). In rodents, acute parenteral elevation of circulating fatty acids has been shown to inhibit insulin signaling via protein kinase C pathways and reduce insulin-stimulated insulin receptor substrate tyrosine phosphorylation (11). In multiple regression analysis in the smaller group for which insulin-infused NEFA levels were available, insulininfused NEFAs displaced fasting NEFAs as an independent factor in insulin sensitivity. In type 2 diabetic subjects, insulin-infused plasma free fatty acids have been shown to correlate strongly with insulin resistance ( 32 ), and from our data there is a similar strength of association in nondiabetic men. Basal and insulin-infused NEFAs were closely associated by linear regression, but also appear to have independent influences on insulin resistance. Elevated insulininfused NEFA levels may reflect a reduced capacity to suppress lipolysis after a physiological increase in insulin, such as in the postprandial state, with increased exposure of skeletal muscle, liver, and pancreas to fatty acids ( 33 ). Limb balance studies, during fasting and insulin-stimulated conditions, reported obese subjects to have reduced levels of basal skeletal muscle fat oxidation compared with lean insulin-sensitive humans ( 12 ). Our whole body indirect calorimetry data would appear to be at variance with this. In our group, insulin resistance was associated with higher rates of basal whole body fat oxidation, in accordance with a Randle effect ( 34 ), that is, fatty acids competing with glucose as substrates for fuel oxidation. Previous studies using whole body calorimetry reported increased basal fat oxidation in obese nondiabetic and glucose-intolerant subjects ( 35 ). There is recent evidence that whole body fat oxidation may be significantly lower only in extremely obese, compared with normal and moderately obese, humans ( 13 ). In our study, however, basal fat oxidation was not an independent predictor of insulin sensitivity. Insulin-resistant humans have reduced suppression of whole body fat oxidation with insulin infusion, which we have previously demonstrated to be an acquired defect ( 14 ). The current study confirms an association between reduced suppression of insulin-infused fat oxidation and insulin resistance. In the whole group, insulin-infused fat oxidation was an independent predictor of insulin sensitivity. However, when insulin-infused NEFA levels were entered into the analysis (although insulin-infused NEFAs were not available for all subjects), insulin-infused fat oxidation was no longer an independent predictor of insulin sensitivity. This may suggest that it is the level of circulating fatty acids that is determining the rate of fat oxidation and is a major influence on insulin sensitivity. Experimental evidence supports this interpretation, with parenteral infusion of lipid/heparin acutely elevating circulating fatty acids and increasing rates of insulin-infused fat oxidation ( 10 ). Ferrannini et al. ( 36 ) showed, in a large group of men and women, that insulin action declines with age, but that when adjusted for BMI, this relationship is no longer significant. In nonobese women ( 37 ), visceral fat increases with increasing age, but explains only a modest part of the decline in insulin sensitivity and was more strongly associated with unfavorable changes in plasma lipids. Our data showed a significant association between increased age and insulin resistance in men and an association between age and central fat (but not total body fat or BMI). An original finding from our data is the strong correlation of age with circulating NEFAs, which may contribute to the association of age with insulin sensitivity. Physical activity is well known to influence insulin sensitivity, both independently and by its influence on visceral fat ( 38 ). Although our group of men was generally sedentary, physical activity was not formally assessed, and differing physical activity levels across the age group is likely to have influenced the association of age and insulin sensitivity. The associations between insulin resistance and hyperlipidemia have been extensively examined. The relative importance of different circulating lipids in predicting cardiovascular disease and their association with insulin resistance are still under investigation ( 39, 40 ). From our data, non-HDL cholesterol or LDL cholesterol were independent predictors of insulin sensitivity, while serum TG, HDL-cholesterol, TG: HDL ratio and total apoB were not. This may be because a rise in serum TG and fall in HDL-cholesterol occur later in the metabolic syndrome and are classically associated with diabetic dyslipidemia (41). In our study, non-HDL cholesterol had a significant inverse association with insulin sensitivity and in multiple regression analysis was an independent negative predictor of insulin sensitivity. Non-HDL cholesterol had a weaker association with central fat, which was not significant when the two subjects with lowest central fat were excluded. NonHDL was also unrelated to circulating NEFA levels, which supports its independent association with insulin sensitivity. Non-HDL cholesterol encompasses LDL cholesterol, but also incorporates possible elevated levels of TG-rich remnants, which may possess cardiovascular risk in addition to that associated with LDL cholesterol ( 42 ). Elevated non-HDL cholesterol also takes into account elevated TG and low HDL cholesterol, and in population studies has been associated with the metabolic syndrome and visceral obesity ( 43 ). An association between insulin resistance and LDL cholesterol levels is less well established. In women, increased LDL cholesterol has been shown to be closely related to increased amounts of central fat, but not to total body fat ( 37 ). In our group of men, although there was a significant association between insulin resistance and LDL cholesterol, the association between LDL cholesterol and central fat was weaker and, as with non-HDL cholesterol, was dependent on the subjects with low central fat. The mechanism of the relationship between LDL cholesterol and insulin sensitivity remains unclear, but is potentially important. Insulin-resistant men are reported to have increased cholesterol synthesis (albeit using an indirect measure of cholesterol synthesis, plasma lathosterol levels) ( 44 ). An increased supply of circulating fatty acids to the liver may have a role in increased VLDL levels and therefore possibly a contributory role in increased LDL cholesterol levels ( 45 ). However, in our study, LDL cholesterol was not associated with increased circulating NEFA levels. Dysregulation of sterol regulatory binding protein-1c function or expression, which has a relationship to cholesterol and TG synthesis, may provide a link between cholesterol metabolism and insulin resistance. Common single nucleotide polymorphisms in the sterol regulatory binding protein-1c gene have been significantly associated with type 2 diabetes in a male Caucasian population ( 46 ). In a different male population, the same allele was associated with significantly higher total and LDL cholesterol levels, but not with 30-min insulin levels or 2-h glucose levels, on oral glucose tolerance testing ( 46 ). LDL cholesterol levels are strongly influenced by clearance rates. In Pima Indians with type 2 diabetes, LDL cholesterol clearance was reduced, although changes in LDL cholesterol levels were minimal ( 47 ). This study used the accepted gold standard assessment of whole body insulin sensitivity, the euglycemic-hyperinsulinemic clamp, to assess the independent predictive value of circulating fatty acids, lipids, and fat oxidation on insulin sensitivity in a relatively large group of men with a wide range of insulin sensitivity and adiposity. Non-HDL cholesterol was an independent predictor of insulin sensitivity, and LDL cholesterol was also negatively associated with insulin sensitivity. However, the mechanism of this relationship is an area that requires additional study. Fasting circulating NEFA levels, although associated with central fat, were an independent predictor of insulin sensitivity. In the group as a whole, reduced suppression of fat oxidation with insulin infusion also predicted insulin resistance. When insulininfused circulating NEFAs are measured, they independently predict insulin sensitivity, displacing both fasting NEFAs and insulin-infused fat oxidation. 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Poynten, A. M., Gan, S. K., Kriketos, A. D., Campbell, L. V., Chisholm, D. J.. Circulating Fatty Acids, Non-High Density Lipoprotein Cholesterol, and Insulin-Infused Fat Oxidation Acutely Influence Whole Body Insulin Sensitivity in Nondiabetic Men, The Journal of Clinical Endocrinology & Metabolism, 2005, 1035-1040, DOI: 10.1210/jc.2004-0943