Conjugated linoleic acid supplementation, insulin sensitivity, and lipoprotein metabolism in patients with type 2 diabetes mellitus

¹ From the Nutrigenomics Research Group, Department of Clinical Medicine, Trinity Centre for Health Sciences, Dublin (FM, AM, and HMR), and the Metabolic Research Unit, St James’s Hospital, Dublin (T-PY and JJN).

² Supported by the Wellcome Trust, United Kingdom.

³ Address reprint requests to HM Roche,Trinity Centre for Health Sciences, St James’s Hospital, James’s Street, Dublin 8, Ireland. E-mail: hmroche{at}tcd.ie.

ABSTRACT  
Background: Some animal studies have suggested that conjugated linoleic acid (CLA) supplementation may have therapeutic potential with respect to insulin sensitivity and lipid metabolism, which are important cardiovascular disease (CVD) risk factors associated with type 2 diabetes mellitus.

Objective: We investigated the effect of CLA supplementation on markers of glucose and insulin metabolism, lipoprotein metabolism, and inflammatory markers of CVD in subjects with type 2 diabetes.

Design: The study was a randomized, double-blind, placebo-controlled trial. Thirty-two subjects with stable, diet-controlled type 2 diabetes received CLA (3.0 g/d; 50:50 blend of cis-9,trans-11 CLA and trans-10,cis-12 CLA) or control for 8 wk. A 3-h 75-g oral-glucose-tolerance test was performed, and fasting plasma lipid concentrations and inflammatory markers were measured before and after the intervention.

Results: CLA supplementation significantly increased fasting glucose concentrations (6.3%; P < 0.05) and reduced insulin sensitivity as measured by homeostasis model assessment, oral glucose insulin sensitivity, and the insulin sensitivity index (composite) (P = 0.05). Total HDL-cholesterol concentrations increased by 8% (P < 0.05), which was due to a significant increase in HDL₂-cholesterol concentrations (P < 0.05). The ratio of LDL to HDL cholesterol was significantly reduced (P < 0.01). CLA supplementation reduced fibrinogen concentrations (P < 0.01) but had no effect on the inflammatory markers of CVD (C-reactive protein and interleukin 6).

Conclusions: CLA supplementation had an adverse effect on insulin and glucose metabolism. Whereas CLA had positive effects on HDL metabolism and fibrinogen, a therapeutic nutrient should not be associated with potentially adverse effects on other clinical markers of type 2 diabetes.

Key Words: Conjugated linoleic acid • diabetes • type 2 diabetes mellitus • humans • metabolic syndrome • cardiovascular disease • glucose • insulin sensitivity • lipoprotein metabolism • inflammation

INTRODUCTION  
In parallel with the global epidemic of type 2 diabetes are significant health and socioeconomic burdens (1). Thus, there is a need to identify effective dietary strategies to attenuate the effect of type 2 diabetes, which is a heterogeneous disease characterized by target-tissue insulin resistance that cannot be overcome by ß cell hypersecretion (2). The World Health Organization (WHO) defined the clustering of metabolic abnormalities associated with type 2 diabetes as the metabolic syndrome. Features include impaired insulin sensitivity; glucose intolerance, or diabetes mellitus coupled with 2 metabolic derangements, including obesity, insulin resistance, hypertension, dyslipidemia (increased plasma triacylglycerol and low HDL-cholesterol concentrations); and microalbuminuria (3). Low-grade inflammation, also a feature of type 2 diabetes (4), has been implicated in the development of atherosclerosis (5). Cardiovascular disease (CVD) is the leading cause of morbidity and mortality among type 2 diabetes patients, as a result of the presence of several primary risk factors for CVD in this patient population (6).

Although little can be done to avert a genetic predisposition to type 2 diabetes, attenuation of the effect of modifiable risk factors through dietary and lifestyle factors is important. Conjugated linoleic acid (CLA) has received attention as a potential therapeutic nutrient with respect to insulin resistance and hyperlipidemia (7), which are key characteristics of type 2 diabetes. The term CLA refers to the positional and geometric isomers of linoleic acid with a conjugated double-bond system (8). Animal feeding studies showed that CLA reduced body fat, increased lean body mass (9–11), improved plasma lipid metabolism, and inhibited the progression and promoted the regression of atherosclerosis (12–14). There are relatively few human intervention studies, and the results of those few are mixed. Most of the evidence regarding body composition suggests that CLA supplementation does not reduce body weight or body fat or increase fat-free mass in humans (15). Small, nonsignificant reductions in LDL cholesterol (16, 17) and conflicting effects regarding HDL cholesterol (16, 18, 19) have been observed after CLA supplementation in humans. A recent study indicated that trans-10,cis-12 CLA (t10,c12 CLA) supplementation had negative effects on insulin resistance and biomarkers of oxidative stress and inflammation in obese men who had signs of the metabolic syndrome (20, 21). In contrast, our group found that a supplement combining cis-9,trans-11 (c9,t11) and t10,c12 CLA improved plasma triacylglycerol concentrations and VLDL metabolism, without adverse effects on insulin and glucose metabolism, in healthy subjects (17). These conflicting results may reflect differences in study duration, cohort composition, study settings, and, most important, supplement composition. CLA is a very heterogenous compound, and distinct isomer-specific effects have been identified (7, 22). The 2 principal CLA isomers, c9,t11 and t10,c12 CLA, have contrasting metabolic and molecular effects. Feeding c9,t11 CLA improved lipid and glucose metabolism, whereas feeding t10,c12 CLA promoted insulin resistance and dylipidemia in ob/ob mice (7, 22).

The objective of the present study was to determine the effect of CLA supplementation (providing equal proportions of c9,t11 and t10,c12 CLA) in patients with stable, diet-controlled type 2 diabetes. To date no study has investigated the metabolic effects of CLA supplementation in these patients. Several key aspects of the metabolic syndrome, including insulin and glucose metabolism, lipoprotein metabolism, and markers of coagulation and inflammation, were measured to ascertain the effect of CLA in persons with diet-controlled type 2 diabetes.

SUBJECTS AND METHODS  
This study was approved by the Joint Ethics Committee of St James’s Hospital and the Federated Dublin Voluntary Hospitals, Ireland. The purpose, nature, and potential risks of the study were explained before written informed consent was obtained from each volunteer. Sample size was estimated by the ability to detect a 20–30% change in triacylglycerol concentrations, assuming a type I error of 0.05 and a power of 0.9. Thirty-two subjects with type 2 diabetes who attended the Diabetic Day Care Centre at St James’s Hospital in Dublin and whose diabetes was controlled by diet therapy alone completed the trial. All subjects had stable metabolic control with a glycated hemoglobin (Hb A_1c) concentration of 6.83 ± 0.18 mmol/L and a mean fasting blood glucose concentration of 7.33 ± 0.24 mmol/L. No subjects were receiving pharmacologic treatment for glucose control or lipid-lowering purposes. Hypertension was present in approximately one-half of the subjects. However, only subjects whose blood pressure was under stable control participated in the study. There was no difference in the prevelance of hypertension between the 2 groups. In addition, there was no documentation of macrovascular disease in the medical notes of any participant. All subjects had stable body weight. All were following healthy eating guidelines as recommended by the American Diabetes Association (23). The study subjects did not consume fatty acid supplements or other dietary products known to affect metabolic markers of type 2 diabetes.

Study design
This randomized, double-blinded, placebo-controlled study was conducted on a free-living, outpatient basis. Subjects received 3.0 g CLA/d (six 0.5-g capsules; a 50:50 isomer blend of c9,t11 and t10,c12 CLA) or placebo (blend of palm oil and soya bean oil) for 8 wk. The placebo was designed to contain a blend of fatty acids that was representative of a habitual Western diet (24, 25). The fatty acid compositions of the CLA supplement and the placebo are shown in Table 1. All supplements were supplied by Loders Crooklann (Wormeveer, Netherlands). Each volunteer received his or her capsules in 2 batches, at baseline and after wk 4. A capsule count was completed midway and at the end of the supplementary period. All participants were asked to maintain their usual dietary and lifestyle habits. The effect of changes in diet, weight, and physical activity was explained to the subjects. There was no change in prescribed medication (ie, antihypertensives) throughout the trial.

View this table:
TABLE 1. Fatty acid composition of the conjugated linoleic acid (CLA) supplement and the control supplement¹

 
Dietary assessment
Mean daily dietary intake was assessed by using two 4-d food records, one completed immediately before the study and the other completed at the end of the supplementation period (26). Household measures, standard food portions, and a food atlas were used to quantify portion sizes (27). This dietary information was analyzed for macronutrient composition with the use of NETWISP software (version 2.0; Tinuviel Software, Warrington, United Kingdom), which was modified to include the composition of frequently consumed foods that were not part of the database. This additional information was obtained from the nutrient information panel supplied by the manufacturers.

Clinical investigations
Subjects attended the Metabolic Unit, Department of Endocrinology, St James’s Hospital, for blood sampling after a 12-h overnight fast before and after intervention (weeks 0 and 8). Subjects abstained from strenuous exercise and alcohol intake for 24 h before the examination and refrained from smoking on the morning of the examination. No medications (eg, antihypertensives) were taken before the clinical investigations. On the morning of each investigation, a fasting blood sample was drawn and a standard 3-h, 75-g oral-glucose-tolerance test (OGTT) was performed. The 75 g anhydrous glucose equivalent (Polycal; Nutricia Clinical, Trowbridge, United Kingdom) was consumed in 300 mL water within 5 min. Blood samples were collected at 0, 30, 60, 90, 120, and 180 min for the measurement of plasma glucose and serum insulin and C-peptide concentrations.

Biochemical analysis
Blood for measurement of cholesterol, triacylglycerol, LDL, interleukin 6 (IL-6), insulin, and C-peptide concentrations was collected in serum tubes (Becton Dickinson, Oxford, United Kingdom). Blood for HDL and VLDL analysis was collected in EDTA-coated evacuated tubes (Becton Dickinson). Samples for glucose analysis were collected in tubes containing sodium fluoride, and blood for fibrinogen, C-reactive protein (CRP), and fatty acid analyses was collected in tubes containing citrate (Becton Dickinson). Serum samples were allowed to clot for 1 h. Samples for lipid, glucose, insulin, C-peptide, and IL-6 analyses were centrifuged immediately at 1400 x g for 10 min at room temperature. Citrated samples for CRP and fibrinogen measurements were collected and then centrifuged at 2000 x g for 20 min at room temperature. Blood samples collected for fatty acid analysis were centrifuged at 200 x g for 20 min at room temperature to harvest the platelet pellet and platelet-poor plasma. All of the samples listed above were frozen immediately and stored at –70°C for subsequent analysis.

Plasma samples were analyzed for triacylglycerol and cholesterol concentrations with the use of enzymatic colorimetric assays supplied by Instrumentation Laboratory (Warrington, United Kingdom) on an IL Monarch centrifugal analyzer (Instrumentation Laboratory) as previously described (28). Total HDL-, HDL₃-, and HDL₂-cholesterol concentrations were determined after precipitation with Immuno Quantolip total HDL precipitation reagent and Immuno Quantolip HDL₃ precipitation reagent (both: Immuno Ag, Vienna) as previously described (17). LDL-cholesterol concentrations were measured after precipitation of LDL by using a Randox precipitation reagent (Randox, Antrim, United Kingdom) according to the manufacturer’s instructions. VLDL was isolated according to published laboratory methods (17). VLDL, LDL, and HDL lipid compositions were determined according to published methods (28). VLDL apolipoprotein B (apoB) was measured by using a turbidimetric assay (Instrumentation Laboratory) on the IL Monarch centrifugal analyzer (29).

Plasma glucose concentrations were measured by using enzymatic determination based on the glucose oxidase principle (BioMerieux, Marcy-l’Etoile, France). Serum insulin and C-peptide concentrations were measured by using solid-phase, two-site fluoroimmunometric assays (AutoDELFIA Insulin kit and AutoDELFIA C-Peptide kit, respectively; Wallac Oy, Turku, Finland) on a 1235 AutoDELFIA automatic immunoassay system (Wallac Oy). Hb A_1c was assessed by using a fully automated HPLC analyzer (Menarini-Arkray HA 8140; Arkray KDK, Kyoto, Japan).

IL-6 and CRP assays were performed by using human high-sensitivity immunoassay test kits (Biosource, Camarillo, CA, and BioCheck Inc, Burlingame, CA, respectively) according to each manufacturer’s instructions. Fibrinogen clotting activity was measured by using an automated clotting assay as previously described (30).

For each marker, both preintervention and postintervention samples for each subject were analyzed within a single batch. The interassay CV for total cholesterol and triacylglycerol was 1.25% and 3.15%, respectively. The interassay CV for fibrinogen was 3.25%. Insulin, C-peptide, Hb A_1c, glucose, and microalbumin concentrations were analyzed in the laboratory of St James’s Hospital and were acceptable according to the routine internal and external standards applied.

Fatty acid compositional analysis
Protocol compliance was verified by conducting a capsule count and by measurement of plasma fatty acid composition with the use of gas liquid chromatography (GLC). Total plasma lipids were isolated by using the method of Folch et al (31), as previously described (17). Methyl esters of total plasma lipid were prepared by adding 0.5 mL of 0.01 mmol NaOH/L in dry methanol and then adding 0.5 mL boron trifluoride as described previously (17). Total plasma lipid fatty acid composition was determined by using Shimadzu GC-14A GLC (Mason Technologies, Dublin) fitted with a Shimadzu C-16A integrator (Mason Technologies) and a CP Sil 88 fused silica column (50 m x 0.22 mm, 0.2 µm file thickness; Chrompack Ltd, Middelburg, Netherlands). Conditions for GLC analysis were as follows: N₂ was the carrier gas, and the column initial temperature (180°C) was increased (at a rate of 5°C/min) to 195°C, held for 40 min, and then increased (by 2°C/min) to 220°C and held for 20 min. Plasma fatty acids were identified according to their retention times in comparison with a fatty acid methyl ester standard (Sigma-Aldrich, Dublin) spiked with known concentrations of transmethylated c9,t11 and t10,c12 CLA isomers (Cayman Chemical, Ann Arbor, MI). Plasma fatty acid composition was calculated as a percentage of total fatty acids.

Anthropometric measurements
Body weight was measured on an electronic balance (to the nearest 0.1 kg) with subjects wearing light clothing but no shoes. Height was assessed by using a stadiometer that measured to the nearest 0.1 cm. Waist girth was measured at the minimum circumference between the iliac crest and the rib cage. Hip girth was measured at the maximum width over the greater trochanters. Waist-to-hip ratio (WHR) was calculated from these measurements (32). Bioelectric impedance analysis was measured in subjects in the erect position by using a body fat analyzer (TBF-300; Tanita Corporation, Arlington Heights, IL). Percentage body fat was calculated by using the manufacturer’s programmed equations.

Data preparation and statistical analysis
All statistical analysis was conducted by using DATA DESK software (version 6.0; Data Description Inc, Ithaca, NY). Results are presented as mean (± SEM). When necessary, values were transformed to give a normal Gaussian distribution. The postprandial data were expressed in summary form—ie, area under the postprandial curve (AUC), incremental area under the postprandial curve (IAUC), maximum postprandial concentration (C_max), and time to maximal postprandial concentration (T_max). These data were used to investigate postprandial variations between the supplement groups. AUC was calculated by using the trapezium rule (33), and IAUC was calculated as the total incremental AUC according to Le Floch (34). Homeostasis model assessment (HOMA) was calculated as fasting glucose (mg/dL) multiplied by fasting insulin (µU/mL) divided by 22.5 (35). Quantitative insulin sensitivity check index (QUICKI) was calculated as 1/[log(I₀) + log(G₀)], where I = insulin and G = glucose (36). The insulin sensitivity index (ISI) composite was calculated as proposed by Matsuda et al (37). Oral glucose insulin sensitivity (OGIS) was calculated by using the published formula (38).

Analysis of covariance (ANCOVA), after control for the baseline value of the outcome variable, was used to identify significant changes in biochemical values after the supplementation period. We controlled for baseline microalbumin and cholesterol concentrations because baseline values differed between the groups. Three-way analysis of variance (ANOVA) with subject, treatment, and intervention as independent variables and a treatment-by-intervention interaction was used to identify significant changes in plasma fatty acid composition. Repeated-measures ANOVA with a treatment-by-time interaction was used to investigate significant differences in postprandial responses between the study groups. Post hoc statistical analysis was conducted by using Scheffe’s test. A P value < 0.05 was considered significant.

RESULTS  
Subject characteristics and intervention details
Baseline characteristics of the 2 study groups are summarized in Table 2. The mean age, weight, body mass index (BMI; in kg/m²), waist circumference, hip circumference, WHR, percentage body fat, and duration of diabetes did not differ significantly between groups. The anthropometric measurements did not change after either the CLA or control supplements (data not shown). Study compliance was assessed by a pill count, total plasma fatty acid composition, and dietary assessment. The subjects consumed 96.16% of the prescribed supplements. Compliance did not differ significantly between the study groups: 96.33% and 95.83% of supplements were used by the control and CLA groups, respectively. The fatty acid compositions of total plasma lipids are shown in Table 3. The c9,t11 CLA concentrations increased by 89% after CLA supplementation (P < 0.001). The t10,c12 CLA isomer was undetectable in most samples and was not affected by CLA supplementation. Linoleic acid (18:2n–6) concentrations increased significantly in the CLA group (P < 0.01). Dietary analysis, based on two 4-d food records, one completed before and the other completed after the intervention, showed that mean daily energy, macronutrient, dietary fiber, cholesterol, alcohol intake, and percentage contributions to total or food energy did not differ significantly after CLA or placebo supplementation (data not shown). In addition, mean daily nutrient intakes did not differ significantly between groups before or after intervention (data not shown).

View this table:
TABLE 2. Baseline characteristics of the study population by treatment group¹

 

View this table:
TABLE 3. Fatty acid composition of total plasma lipids before and after conjugated linoleic acid (CLA) supplementation or placebo for 8 wk¹

 
Insulin and glucose metabolism
The effect of CLA supplementation on a range of measures of insulin sensitivity and glycemic control is shown in Table 4. Fasting glucose concentrations were significantly increased after CLA supplementation (P < 0.05). HOMA was also significantly higher after CLA supplementation than after control supplementation (P = 0.05). Both OGIS and the ISI (composite) were significantly lower after CLA supplementation than after control supplementation (P = 0.05 and P < 0.05, respectively). The QUICKI measure of insulin sensitivity and the Hb A_1c and microalbumin concentrations were not significantly altered by CLA supplementation. The postprandial glucose, insulin, and C-peptide concentrations in response to the OGTT are shown in Figure 1. Repeated-measures ANOVA showed that plasma glucose, serum insulin, and C-peptide concentrations increased significantly (P < 0.001) during the OGTT. Both before and after the intervention, postprandial glucose concentrations were significantly higher in the CLA group, whereas postprandial C-peptide concentrations were significantly higher in the control group (P < 0.01). Fasting glucose concentrations were significantly higher after CLA supplementation than after control supplementation (P < 0.05). Postprandial glucose concentrations were not significantly changed by either supplement. In addition, neither supplement had a significant effect on fasting or postprandial insulin or C-peptide concentrations. Summary variables (AUC, IAUC, T_max, C_max) of the postprandial glucose and insulin response after the OGTT were not significantly altered by CLA or control supplementation (data not shown).

View this table:
TABLE 4. Measurements of insulin sensitivity and glycemic control before and after conjugated linoleic acid (CLA) supplementation or placebo for 8 wk¹

 

View larger version (16K):
FIGURE 1.. Mean (± SEM) plasma glucose, serum insulin, and C-peptide concentrations in response to the administration of 75 g anhydrous glucose before and after conjugated linoleic acid (CLA) supplementation (3 g/d) or placebo for 8 wk in the control group (week 0, ; week 8, ) and the CLA group (week 0, •; week 8, ). By repeated-measures ANOVA, there was a significant time effect for glucose, insulin, and C-peptide (P < 0.001) and a treatment-by-time effect for plasma glucose and serum C-peptide (P < 0.01). Fasting plasma glucose concentrations were significantly higher after CLA supplementation, P < 0.05 (analysis of covariance).

 
Lipoprotein metabolism
Fasting serum and lipoprotein lipid concentrations before and after supplementation are shown in Table 5. There was no significant effect of either CLA or control supplementation on total or LDL-cholesterol concentrations. Total HDL-cholesterol concentrations were significantly increased after CLA supplementation (P < 0.05). The 7.9% increase in total HDL-cholesterol concentration was due to a significant increase in HDL₂ concentrations after CLA supplementation (P < 0.05). LDL:HDL cholesterol was significantly reduced after CLA supplementation (P < 0.01). Serum and VLDL triacylglycerol and VLDL-cholesterol concentrations were not significantly altered by either supplement. VLDL apoB concentrations were significantly higher after CLA supplementation than after control supplementation (P < 0.05). Triacylglycerol-poor lipoprotein triacylglycerol and cholesterol concentrations were not significantly altered by the CLA or placebo supplements (data not shown).

View this table:
TABLE 5. Mean fasting plasma and lipoprotein cholesterol and triacylglycerol concentrations before and after conjugated linoleic acid (CLA) supplementation or placebo for 8 wk¹

 
Markers of inflammation and coagulation
The effect of CLA and control supplementation on inflammation and coagulation was also determined. Plasma fibrinogen concentrations were significantly lower after CLA supplementation than after control supplementation (315.11 ± 7.34, 317.36 ± 5.56, 310.15 ± 5.84, and 294.30 ± 5.87 mg/dL for control week 0, control week 8, CLA week 0, and CLA week 8, respectively) significantly more than did control supplementation (P < 0.01). Serum IL-6 and plasma CRP concentrations were not altered by CLA or control supplements (data not shown).

DISCUSSION  
This study determined the effect of CLA supplementation on several key CVD risk factors in a group of healthy subjects with well-controlled type 2 diabetes that was treated by diet therapy alone. In view of the increased risk of CVD associated with diabetes, it is important to investigate the ability of nutritional therapies to attenuate the effect of modifiable risk factors associated with CVD in this high-risk patient group. Glucose tolerance and insulin sensitivity are central to type 2 diabetes, and therefore an OGTT was performed, and a number of fasting and postprandial indexes were investigated. CLA supplementation had a negative effect on fasting glucose concentrations. HOMA is a common measure of insulin resistance, derived from fasting insulin and glucose concentrations. In our study, CLA supplementation had an adverse effect on insulin resistance, increasing HOMA by 19%. Postprandial plasma glucose concentration has been cited as an independent risk factor for CVD in persons with type 2 diabetes (39). Hepatic and peripheral insulin sensitivities differ considerably within subjects, and a combination of fasting and postprandial measures provides a more comprehensive measure of insulin sensitivity than does either measure alone (37). Postprandial glucose and insulin concentrations did not increase significantly after CLA supplementation, but the ISI (composite), which was derived from both fasting and postprandial measures, decreased significantly after CLA supplementation. In addition, OGIS, which is also based on both fasting and postprandial data, was significantly reduced by CLA supplementation. It has been proposed that OGIS represents a better index of insulin sensitivity because it has a more physiologic basis than the empirical formulas have (38). Moreover, OGIS strongly correlates with the hyperinsulinemic-euglycemic glucose clamp measures of insulin sensitivity in diabetic subjects, whereas HOMA and ISI (composite) do not (38).

This study investigated the effect of a CLA supplement that provides equal proportions of the c9,t11 and t10,c12 CLA isomers, the composition of which reflects most commercially available CLA supplements. It is interesting that this supplement did not adversely affect insulin and glucose metabolism in healthy subjects (17). The present study confirms work that showed that t10,c12 CLA supplementation had a negative effect on insulin resistance in obese men with signs of the metabolic syndrome (20). The discrepancy between studies reflects the diverse isomer-specific metabolic and molecular effects of CLA. Our group found that feeding a t10,c12 CLA–rich diet induced a profound prodiabetic effect, whereas feeding the c9,t11 CLA isomer improved lipid and glucose metabolism in ob/ob mice (7, 22). Other groups have shown that feeding a blend of CLA isomers induced marked lipodystrophic insulin resistance and glucose intolerance in the C57BL/6J mouse model of diabetes (40). In contrast, another group showed that feeding a 50:50 blend of c9,t11 and t10,c12 CLA reduced glucose and insulin concentrations in male ZDF rats (41, 42). Clearly the effect of CLA supplementation is isomer-specific and dependent on the diabetic risk of the experimental model. In the case of subjects with type 2 diabetes, a CLA blend containing equal quantities of c9,t11 and t10,c12 CLA does not improve insulin and glucose metabolism.

CLA supplementation had a positive effect on HDL-cholesterol concentrations in this study. It has been reported that, for each increase of 0.03 mmol/L in HDL-cholesterol concentration, there is a 2–3% reduction in coronary heart disease (CHD) risk (43). Thus, the increase in HDL cholesterol after CLA supplementation could represent a 7.3–11% reduction in CHD risk. The increased HDL₂-cholesterol concentrations could promote reverse cholesterol transport (RCT; 44). Because RCT is impaired in persons with type 2 diabetes (45), the potential ability of CLA to restore RCT would be favorable. CLA supplementation reduced LDL-cholesterol concentrations (8.8%), albeit not significantly, but that reduction contributed to a greater reduction (14.5%) in LDL:HDL cholesterol. The Helsinki Heart Study showed that LDL:HDL cholesterol was the single best predictor of cardiac events (46). Therefore, the effect of CLA supplementation on LDL:HDL cholesterol in type 2 diabetes may be of clinical benefit.

The lack of effect on CLA on triacylglycerol metabolism contrasts with our previous findings (17). It is possible that this patient cohort may be resistant to CLA-induced improvements in triacylglycerol metabolism, an effect that may be linked to the adverse effects of CLA on glucose and insulin metabolism. VLDL apoB concentrations increased after CLA supplementation. Smedman et al (47) also showed an increase in plasma apoB concentrations after CLA supplementation. Increased intrahepatic lipid substrate (triacylglycerol and cholesterol) delivery is a critical factor regulating hepatic VLDL apoB secretion. However, neither triacylglycerol nor cholesterol concentrations were significantly altered after CLA supplementation. Increased VLDL apoB concentrations are associated with insulin resistance (48). Therefore, the increase in VLDL apoB concentrations in this study may be related to increased insulin resistance.

Elevated plasma concentrations of fibrinogen, CRP, and IL-6 are associated with increased risk of CHD and the severity of atherosclerosis (49). Both low-grade inflammation (4) and a prothrombotic risk profile have been reported in persons with type 2 diabetes (50). CLA supplementation reduced plasma fibrinogen concentrations, which have a key role in coagulation (51). CRP concentrations showed a nonsignificant reduction (15%) after CLA supplementation. A previous study showed that CLA supplementation had no effect on markers of blood coagulation and platelet function in a small group of healthy women who were given a CLA supplement containing a mixture of 10 CLA isomers for 63 d (18). This isomeric blend is in contrast to our supplement. CRP concentrations were significantly increased in obese men with signs of the metabolic syndrome (21). The latter result may reflect the isomer-specific effect of t10,c12 CLA. CRP stimulates tissue factor production, which is the main stimulus for initiating coagulation (49). Therefore, reduced plasma fibrinogen and CRP concentrations indicate that CLA may attenuate thrombosis.

Baseline plasma concentrations of c9,t11 CLA and the percentage increase after CLA supplementation are comparable to those in previous supplementation studies (17, 52, 53). In contrast, there were negligible concentrations of t10,c12 CLA in baseline samples, and they did not increase after CLA supplementation, despite the fact that the supplement provided t10,c12 CLA. The difficulty in detecting the t10,c12 CLA isomer has been documented previously (17). Two hypotheses for this observation have been suggested. First, the t10,c12 CLA may be more easily oxidized because of its structure, and this would allow it to bypass a number of rate-limiting steps in the peroxisomal ß-oxidation pathway (54). Alternatively, Sebedio et al (55) showed that the t10,c12 CLA isomer is metabolized into 20:4 5,8,12,14 and 20:3 8,12,14 via desaturation and elongation pathways. Consequently, t10,c12 CLA is not efficiently incorporated into plasma lipids.

In conclusion, CLA supplementation did not have a positive effect on insulin and glucose concentrations in persons with type 2 diabetes. Whereas we did show positive effects of CLA on HDL-cholesterol and fibrinogen concentrations, the relative importance of one CVD risk factor over another is unknown. Compliance was ensured, given the significant increase in c9,t11 CLA in total plasma lipid fraction, which was comparable with previously reported data (17, 52). Any truly effective dietary strategy would improve CVD risk factors without any negative effect on other components of the metabolic syndrome.

ACKNOWLEDGMENTS  
We are indebted to the study participants for their enthusiasm and commitment to the study protocol and to Kim Jackson for her aid in the lipoprotein analysis.

FM prepared the manuscript and conducted most of the experiments. TPY conducted the clinical investigations. AM assisted in the experimental work. JJN was involved in the formulation of the scientific hypothesis for this study and provided significant advice. HMR formulated the scientific hypothesis and experimental design and prepared the manuscript. None of the authors have any commercial interest in CLA, and none of the authors had any other conflict of interest.

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