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Conjugated linoleic acid supplementation, insulin sensitivity, and
lipoprotein metabolism in patients with type 2 diabetes mellitus
1–3
Fiona Moloney, Toh-Peng Yeow, Anne Mullen, John J Nolan, and Helen M Roche
ABSTRACT
Background: Some animal studies have suggested that conjugated
linoleicacid(CLA)supplementationmayhavetherapeuticpotential
with respect to insulin sensitivity and lipid metabolism, which are
importantcardiovasculardisease(CVD)riskfactorsassociatedwith
type 2 diabetes mellitus.
Objective: We investigated the effect of CLA supplementation on
markersofglucoseandinsulinmetabolism,lipoproteinmetabolism,
and inflammatory markersof CVD in subjects with type 2 diabetes.
Design: The study was a randomized, double-blind, placebo-
controlledtrial. Thirty-two subjects withstable, diet-controlled type
2diabetesreceivedCLA(3.0g/d;50:50blendofcis-9,trans-11CLA
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 con-
centrations and inflammatory markers were measured before and
after the intervention.
Results: CLA supplementation significantly increased fasting glu-
coseconcentrations(6.3%;P쏝 0.05) andreducedinsulinsensitivity
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 쏝 0.05),
which was due to a significant increase in HDL
2
-cholesterol con-
centrations (P 쏝 0.05). The ratio of LDL to HDL cholesterol was
significantly reduced (P 쏝 0.01). CLA supplementation reduced
fibrinogen concentrations (P 쏝 0.01) but had no effect on the in-
flammatory markers of CVD (C-reactive protein and interleukin 6).
Conclusions: CLA supplementation had an adverse effect on insu-
lin and glucose metabolism. Whereas CLA had positive effects on
HDL metabolism and fibrinogen, a therapeutic nutrient should not
beassociatedwithpotentially adverse effectsonotherclinicalmark-
ers of type 2 diabetes. Am J Clin Nutr 2004;80:887–95.
KEY WORDS Conjugated linoleic acid, diabetes, type 2 dia-
betes mellitus, humans, metabolic syndrome, cardiovascular dis-
ease, glucose, insulin sensitivity, lipoprotein metabolism, inflam-
mation
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 char-
acterized by target-tissue insulin resistance that cannot be over-
come by

cell hypersecretion (2). The World Health Organiza-
tion (WHO) defined the clustering of metabolic abnormalities
associated with type 2 diabetes as the metabolic syndrome. Fea-
turesincludeimpairedinsulinsensitivity;glucoseintolerance,or
diabetes mellitus coupled with 욷2 metabolic derangements, in-
cluding obesity, insulin resistance, hypertension, dyslipidemia
(increasedplasmatriacylglyceroland low HDL-cholesterolcon-
centrations); and microalbuminuria (3). Low-grade inflamma-
tion, 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
type2diabetes,attenuationoftheeffectofmodifiableriskfactors
through dietary and lifestyle factors is important. Conjugated
linoleic acid (CLA) has received attention as a potential thera-
peutic nutrient with respect to insulin resistance and hyperlipid-
emia (7), which are key characteristics of type 2 diabetes. The
term CLA refers to the positional and geometric isomers of lino-
leic 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 athero-
sclerosis (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 sup-
plementationdoesnotreducebodyweightorbodyfatorincrease
fat-freemassinhumans(15).Small,nonsignificantreductionsin
LDL cholesterol (16, 17) and conflicting effects regarding HDL
cholesterol (16, 18, 19) have been observed after CLA supple-
mentation 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).Incontrast,ourgroupfoundthatasupplement
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
1
From the Nutrigenomics Research Group, Department of Clinical Med-
icine, 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 Sci-
ences, St James’s Hospital, James’s Street, Dublin 8, Ireland. E-mail:
hmroche@tcd.ie.
Received February 2, 2004.
Accepted for publication April 7, 2004.
887Am J Clin Nutr 2004;80:887–95. Printed in USA. © 2004 American Society for Clinical Nutrition
by guest on June 13, 2013ajcn.nutrition.orgDownloaded from
healthy subjects (17). These conflicting results may reflect dif-
ferences in study duration, cohort composition, study settings,
and, most important, supplement composition. CLA is a very
heterogenous compound, and distinct isomer-specific effects
havebeenidentified(7,22).The2principalCLAisomers,c9,t11
and t10,c12 CLA, have contrasting metabolic and molecular
effects. Feeding c9,t11 CLA improved lipid and glucose metab-
olism,whereasfeedingt10,c12CLApromotedinsulinresistance
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 hasinvestigated the metabolic effects
ofCLAsupplementationinthesepatients.Several key aspectsof
the metabolic syndrome, including insulin and glucose metabo-
lism, 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’sHospital and theFederated Dublin VoluntaryHospitals,
Ireland.Thepurpose,nature,andpotentialrisksofthestudywere
explained before written informed consent was obtained from
eachvolunteer.Samplesizewasestimatedbytheabilitytodetect
a20–30% 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’sHospitalinDublinandwhosediabeteswascontrolledby
diet therapy alone completed the trial. All subjects had stable
metabolic control with a glycatedhemoglobin (HbA
1c
) concen
-
tration of 6.83 앐 0.18 mmol/L and a mean fasting blood glucose
concentration of 7.33 앐 0.24 mmol/L. No subjects were receiv-
ing pharmacologic treatment for glucose control or lipid-
lowering purposes. Hypertension was present in approximately
one-half of the subjects. However, only subjects whose blood
pressurewasunderstablecontrolparticipatedin the study.There
was no difference in the prevelance of hypertension between the
2 groups. In addition, there was no documentation of macrovas-
culardisease in the medical notes of any participant. All subjects
hadstablebodyweight.Allwerefollowing healthy eatingguide-
lines as recommended by the American Diabetes Association
(23). The study subjects did notconsume 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 re-
ceived 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).ThefattyacidcompositionsoftheCLAsupplementand
theplaceboareshowninTable1.Allsupplementsweresupplied
by Loders Crooklann (Wormeveer, Netherlands). Each volun-
teerreceivedhisorhercapsulesin2batches,atbaselineandafter
wk 4. A capsule count was completed midway and at the end of
the supplementary period. All participants were asked to main-
tain their usual dietary and lifestyle habits. The effect of changes
in diet, weight, and physical activity was explained to the sub-
jects. There was no change in prescribed medication (ie, antihy-
pertensives) throughout the trial.
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
wereusedtoquantifyportionsizes(27).Thisdietaryinformation
was analyzed for macronutrient composition with the use of
NETWISP software (version 2.0; Tinuviel Software, War-
rington, 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 Endo-
crinology, 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, antihyper-
tensives) were taken before the clinical investigations. On the
morningofeachinvestigation,afastingblood sample wasdrawn
andastandard 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 flu-
oride, and blood for fibrinogen, C-reactive protein (CRP), and
TABLE 1
Fatty acid composition of the conjugated linoleic acid (CLA) supplement
and the control supplement
1
Ingredient
Supplement
Control CLA
% by wt of total fatty acids
Fatty acid composition (%)
16:0 38.52 4.82
18:0 4.70 2.28
18:1 34.84 10.17
18:2nҀ6 17.89 1.73
Saturated fatty acids 44.97 8.16
CLA isomers (%)
Total CLA 0.06 73.83
cis-9, trans-11 CLA 0.06 35.62
trans-10, cis-12 CLA ND 38.21
1
Supplements were given as free fatty acid. ND, not detectable.
888 MOLONEY ET AL
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fattyacidanalyseswascollectedintubescontainingcitrate(Bec-
ton 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 ҂ g for 10 min at room
temperature. Citrated samples for CRP and fibrinogen measure-
mentswerecollectedandthencentrifugedat2000҂ gfor20min
atroomtemperature.Bloodsamplescollectedfor fatty acidanal-
ysis were centrifuged at 200 ҂ 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
Ҁ70 °C for subsequent analysis.
Plasma samples were analyzed for triacylglycerol and choles-
terol concentrations with the use of enzymatic colorimetric as-
says supplied by Instrumentation Laboratory (Warrington,
United Kingdom) on an IL Monarch centrifugal analyzer (In-
strumentation Laboratory) as previously described (28). Total
HDL-, HDL
3
-, and HDL
2
-cholesterol concentrations were de
-
termined after precipitation with Immuno Quantolip total HDL
precipitationreagentandImmunoQuantolipHDL
3
precipitation
reagent (both: Immuno Ag, Vienna) as previously described
(17). LDL-cholesterol concentrations were measured after pre-
cipitation of LDL by using a Randox precipitation reagent (Ran-
dox, Antrim, United Kingdom) according to the manufacturer’s
instructions. VLDL was isolated according to published labora-
tory 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 centrif-
ugal analyzer (29).
Plasma glucose concentrations were measured by using enzy-
matic 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 immunoas-
say 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
wasmeasuredbyusinganautomatedclottingassayaspreviously
described (30).
For each marker, both preintervention and postintervention
samplesforeachsubjectwereanalyzedwithinasinglebatch.The
interassayCVfortotalcholesterolandtriacylglycerolwas1.25%
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
countandbymeasurementofplasmafattyacidcompositionwith
theuse ofgas liquid chromatography (GLC). Totalplasma lipids
were isolated by using the method of Folch et al (31), as previ-
ously described (17). Methyl esters of total plasma lipid were
prepared by adding 0.5 mL of 0.01 mmol NaOH/L in dry meth-
anol 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 Technol-
ogies, Dublin) fitted with a Shimadzu C-16A integrator (Mason
Technologies) and a CP Sil 88 fused silica column (50 m ҂
0.22 mm, 0.2
m file thickness; Chrompack Ltd, Middelburg,
Netherlands). Conditions for GLC analysis were as follows: N
2
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
nearest0.1 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 cir-
cumferencebetweentheiliaccrestandtheribcage.Hipgirthwas
measured at the maximum width over the greater trochanters.
Waist-to-hip ratio (WHR) was calculated from these measure-
ments (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 pro-
grammed equations.
Data preparation and statistical analysis
All statistical analysis was conducted by using DATA DESK
software(version6.0;DataDescriptionInc,Ithaca,NY).Results
are presented as mean (앐 SEM). When necessary, values were
transformed to give a normal Gaussian distribution. The post-
prandial data were expressed in summary form—ie, area under
the postprandial curve (AUC), incremental area under the post-
prandial curve (IAUC), maximum postprandial concentration
(C
max
), and time to maximal postprandial concentration (T
max
).
These data were used to investigate postprandial variations be-
tween the supplement groups. AUC was calculated by using the
trapezium rule (33), and IAUC was calculated as the total incre-
mental AUC according to Le Floch (34). Homeostasis model
assessment (HOMA) was calculated as fasting glucose (mg/dL)
multiplied by fasting insulin (
U/mL) divided by 22.5 (35). Quan-
titative insulinsensitivity check index (QUICKI) was calculated as
1/[log(I
0
)ѿ log(G
0
)], where I ҃ insulin and G ҃ glucose (36).The
insulinsensitivityindex(ISI)compositewascalculatedasproposed
by Matsuda et al (37). Oral glucose insulin sensitivity (OGIS) was
calculated by using the published formula (38).
Analysisofcovariance(ANCOVA),aftercontrolforthebase-
line value of the outcome variable, was used to identify signifi-
cant changes in biochemical values after the supplementation
period.Wecontrolledforbaselinemicroalbuminandcholesterol
concentrations because baseline values differed between the
groups. Three-way analysis of variance (ANOVA) with subject,
treatment, and intervention as independent variables and a
CLA AND TYPE 2 DIABETES MELLITUS 889
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treatment-by-intervention interaction was used to identify sig-
nificant changes in plasma fatty acid composition. Repeated-
measures ANOVA with a treatment-by-time interaction was
used to investigate significant differences in postprandial re-
sponses between the study groups. Post hoc statistical analysis
was conducted by using Scheffe’s test. A P value 쏝 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/m
2
),waist circumference, hip circumference, WHR, percent
-
age 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 sub-
jects consumed 96.16% of the prescribed supplements. Compli-
ance did not differ significantly between the study groups:
96.33%and95.83%ofsupplementswereusedbythecontroland
CLA groups, respectively. The fatty acid compositions of total
plasma lipids are shown in Table 3. The c9,t11 CLA concentra-
tions increased by 89% after CLA supplementation (P 쏝 0.001).
The t10,c12 CLA isomer was undetectable in most samples and
was not affected by CLA supplementation. Linoleic acid (18:
2nҀ6) concentrations increased significantly in the CLA group
(P 쏝 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 nu-
trient intakes did not differ significantly between groups before
or after intervention (data not shown).
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.
Fastingglucoseconcentrationsweresignificantlyincreasedafter
CLAsupplementation(P쏝 0.05). HOMAwasalsosignificantly
higherafter CLAsupplementation thanafter controlsupplemen-
tation (P ҃ 0.05). Both OGIS and the ISI (composite) were
significantlylowerafterCLA supplementation thanaftercontrol
supplementation (P ҃ 0.05 and P 쏝 0.05, respectively). The
QUICKI measure of insulin sensitivity and the Hb A
1c
and mi
-
croalbumin concentrations were not significantly altered by
CLA supplementation. The postprandial glucose, insulin, and
C-peptide concentrations in response to the OGTT are shown in
Figure1. Repeated-measures ANOVA showed that plasma glu-
cose, serum insulin, and C-peptide concentrations increased sig-
nificantly (P 쏝 0.001) during the OGTT. Both before and after
TABLE 2
Baseline characteristics of the study population by treatment group
1
Control group
(n ҃ 16)
CLA group
(n ҃ 16)
Age (y) 58.1 앐 2.7 63.8 앐 2.2
Weight (kg) 88.2 앐 3.8 84.37 앐 3.1
BMI (kg/m
2
)
30.7 앐 1.2 29.1 앐 1.0
Waist circumference (cm) 101.1 앐 2.6 102.7 앐 2.5
Hip circumference (cm) 110.0 앐 2.8 108.2 앐 1.9
WHR 0.92 앐 0.02 0.95 앐 0.02
Percentage body fat (%) 33.2 앐 2.3 32.9 앐 2.5
Duration of diabetes (mo) 32.9 앐 7.4 34.5 앐 5.74
1
All values are x 앐 SEM. CLA, conjugated linoleic acid; WHR,waist-
to-hipratio. Therewere nosignificantdifferencesinbaselinevaluesbetween
the 2 groups (one-way ANOVA).
TABLE 3
Fatty acid composition of total plasma lipids before and after conjugated linoleic acid (CLA) supplementation or placebo for 8 wk
1
Fatty acid
Control group
(n ҃ 16)
CLA group
(n ҃ 16)
Week 0 Week 8 Week 0 Week 8
% by wt of total fatty acids
16:0 24.80 앐 0.55 25.52 앐 0.58 25.75 앐 0.57 26.3 앐 0.50
16:1 2.16 앐 0.22 1.79 앐 0.13 2.17 앐 0.22 2.24 앐 0.19
18:0 9.96 앐 0.40 10.97 앐 0.55 10.3 앐 0.40 10.78 앐 0.40
18:1 18.58 앐 0.85 17.66 앐 0.80 18.46 앐 0.88 19.66 앐 0.96
18:2nҀ6 20.73 앐 0.72 21.26 앐 0.91 19.21 앐 0.74 21.90 앐 1.18
2
18:3nҀ6 0.26 앐 0.02 0.23 앐 0.01 0.20 앐 0.02 0.24 앐 0.01
18:3nҀ3 0.49 앐 0.08 0.57 앐 0.1 0.44 앐 0.06 0.32 앐 0.05
CLA 0.28 앐 0.04 0.32 앐 0.05 0.37 앐 0.06 0.7 앐 0.08
3
20:2 0.27 앐 0.02 0.29 앐 0.02 0.30 앐 0.01 0.29 앐 0.02
20:3nҀ6 2.52 앐 0.15 2.59 앐 0.24 2.31 앐 0.25 2.30 앐 0.14
20:4nҀ6 8.58 앐 0.37 9.06 앐 0.53 9.36 앐 0.56 8.66 앐 0.46
20:5nҀ3 1.30 앐 0.16 1.24 앐 0.24 1.67 앐 0.32 1.18 앐 0.14
22:4nҀ6 0.41 앐 0.03 0.43 앐 0.06 0.70 앐 0.18 0.40 앐 0.03
22:6nҀ3 2.94 앐 0.25 2.47 앐 0.23 3.15 앐 0.29 3.16 앐 0.26
1
All values are x 앐 SEM.
2
Significantly different from week 0, P 쏝 0.01 (three-way ANOVA).
3
Significant difference between groups after 8 wk of supplementation (treatment ҂ intervention interaction), P 쏝 0.001 (three-way ANOVA).
890 MOLONEY ET AL
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the intervention, postprandial glucose concentrations were sig-
nificantly higher in the CLA group, whereas postprandial
C-peptideconcentrationsweresignificantly higher inthecontrol
group (P 쏝 0.01). Fasting glucose concentrations were signifi-
cantly higher after CLA supplementation than after control sup-
plementation (P 쏝 0.05). Postprandial glucose concentrations
werenotsignificantlychangedbyeithersupplement.Inaddition,
neither supplement had a significant effect on fasting or post-
prandialinsulinorC-peptideconcentrations.Summaryvariables
(AUC,IAUC,T
max
,C
max
)ofthepostprandialglucoseandinsulin
response after the OGTT were not significantly altered by CLA
or control supplementation (data not shown).
Lipoprotein metabolism
Fasting serum and lipoprotein lipid concentrations before and
after supplementation are shown in Table 5. There was no sig-
nificant effect of either CLA or control supplementation on total
or LDL-cholesterol concentrations. Total HDL-cholesterol con-
centrations were significantly increased after CLA supplemen-
tation (P 쏝 0.05). The 7.9% increase in total HDL-cholesterol
concentration was due to a significant increase in HDL
2
concen
-
trations after CLA supplementation (P 쏝 0.05). LDL:HDL cho-
lesterol was significantly reduced after CLA supplementation
(P 쏝 0.01). Serum and VLDL triacylglycerol and VLDL-
cholesterol concentrations were not significantly altered by ei-
ther supplement. VLDL apoB concentrations were significantly
higherafter CLAsupplementation thanafter controlsupplemen-
tation (P 쏝 0.05). Triacylglycerol-poor lipoprotein triacylglyc-
erolandcholesterolconcentrationswerenotsignificantlyaltered
by the CLA or placebo supplements (data not shown).
Markers of inflammation and coagulation
The effect of CLA and control supplementation on inflamma-
tion and coagulation was also determined. Plasma fibrinogen
concentrations were significantly lower after CLA supplemen-
tationthanaftercontrol supplementation (315.11앐 7.34,317.36
앐 5.56, 310.15 앐 5.84, and 294.30 앐 5.87 mg/dL for control
week 0, control week 8, CLA week 0, and CLA week 8, respec-
tively)significantly more than did control supplementation (P 쏝
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 asso-
ciated with CVD in this high-risk patient group. Glucose toler-
ance 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 supple-
mentation 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
andpostprandialmeasuresprovides a morecomprehensivemea-
sure of insulin sensitivity than does either measure alone (37).
Postprandial glucose and insulin concentrations did not increase
significantly after CLA supplementation, but the ISI (compos-
ite), which was derived from both fasting and postprandial mea-
sures, decreased significantly after CLA supplementation. In
addition, OGIS, which is also based on both fasting and post-
prandial data, was significantly reduced by CLA supplementa-
tion. 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 cor-
relates 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 iso-
mers, the composition of which reflects most commercially
availableCLAsupplements. Itisinteresting thatthis 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
TABLE 4
Measurements of insulin sensitivity and glycemic control before and after conjugated linoleic acid (CLA) supplementation or placebo for 8 wk
1
Control group
(n ҃ 16)
CLA group
(n ҃ 16)
Week 0 Week 8 Week 0 Week 8
Fasting glucose (mmol/L) 7.33 앐 0.40 7.09 앐 0.35 7.34 앐 0.27 7.8 앐 0.28
2
Fasting insulin (pmol/L) 54.94 앐 5.10 50.68 앐 7.60 52.13 앐 7.90 57.87 앐 8.86
HOMA 3.00 앐 0.35 2.59 앐 0.42 2.81 앐 0.42 3.35 앐 0.54
3
QUICKI 0.33 앐 0.005 0.36 앐 0.025 0.34 앐 0.007 0.33 앐 0.007
ISI (composite) 3.98 앐 0.40 6.26 앐 1.89 4.58 앐 0.64 4.11 앐 0.54
2
OGIS 353.63 앐 19.0 362.40 앐 16.41 341.08 앐 15.89 329.84 앐 16.38
3
Hb A
1c
6.73 앐 0.12 6.59 앐 0.18 6.93 앐 0.33 6.63 앐 0.16
Microalbumin 3.22 앐 0.77 3.32 앐 0.72 1.61 앐 0.31 1.49 앐 0.21
1
All values are x 앐 SEM. HOMA, homeostasis model assessment; QUICKI, quantitative insulin sensitivity check index; ISI (composite), insulin
sensitivity index (composite); OGIS, oral glucose insulin sensitivity; Hb A
1c
, hemoglobin A
1c
.
2,3
Significant difference between groups after 8 wk of supplementation (analysis of covariance):
2
P 쏝 0.05,
3
P ҃ 0.05.
CLA AND TYPE 2 DIABETES MELLITUS 891
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FIGURE1.Mean(앐 SEM) plasmaglucose,serum insulin,and C-peptideconcentrationsin responseto theadministrationof 75g anhydrousglucosebefore
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, F; week 8, 䉫). By repeated-measures ANOVA, there was a significant time effect for glucose, insulin, and C-peptide (P 쏝 0.001) and a treatment-by-time
effect for plasma glucose and serum C-peptide (P 쏝 0.01). Fasting plasma glucose concentrations were significantly higher after CLA supplementation, P 쏝
0.05 (analysis of covariance).
892 MOLONEY ET AL
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syndrome (20). The discrepancy between studies reflects the
diverseisomer-specificmetabolicandmoleculareffectsofCLA.
Our group found that feeding a t10,c12 CLA–rich diet induced a
profound prodiabetic effect, whereas feeding the c9,t11 CLA
isomerimproved 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-
cholesterolconcentrationsinthis study. Ithasbeen reported that,
for each increase of 0.03 mmol/L in HDL-cholesterol concen-
tration, 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
CHDrisk.TheincreasedHDL
2
-cholesterolconcentrationscould
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 supple-
mentation reduced LDL-cholesterol concentrations (8.8%), al-
beit 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 con-
trasts with our previous findings (17). It is possible that this
patientcohortmayberesistanttoCLA-inducedimprovementsin
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 supplementa-
tion.Smedmanetal(47)alsoshowedanincreaseinplasmaapoB
concentrations after CLA supplementation. Increased intrahe-
patic lipid substrate (triacylglycerol and cholesterol) delivery is
a critical factor regulating hepatic VLDL apoB secretion. How-
ever, neither triacylglycerol nor cholesterol concentrations were
significantly altered after CLA supplementation. Increased
VLDL apoB concentrations are associated with insulin resis-
tance(48).Therefore,theincreaseinVLDLapoBconcentrations
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 fi-
brinogen concentrations, which have a key role in coagulation
(51). CRP concentrations showed a nonsignificant reduction
(15%)afterCLAsupplementation.Apreviousstudyshowedthat
CLA supplementation had no effect on markers of blood coag-
ulation 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 in-
creasedin 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 isthe 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 per-
centage increase after CLA supplementation are comparable to
those in previous supplementation studies (17, 52, 53). In con-
trast, there were negligible concentrations of t10,c12 CLA in
baseline samples, and they did not increase after CLA supple-
mentation, despite the fact that the supplement providedt10,c12
CLA. The difficulty in detecting the t10,c12 CLA isomer has
been documented previously (17). Two hypotheses for this ob-
servation have been suggested. First, the t10,c12 CLA may be
TABLE 5
Mean fasting plasma and lipoprotein cholesterol and triacylglycerol concentrations before and after conjugated linoleic acid (CLA) supplementation or
placebo for 8 wk
1
Control group
(n ҃ 16)
CLA group
(n ҃ 16)
Week 0 Week 8 Week 0 Week 8
Plasma Cholesterol (mmol/L) 4.73 앐 0.26 4.78 앐 0.23 5.16 앐 0.17
2
5.06 앐 0.14
Cholesterol (mmol/L)
LDL 3.10 앐 0.22 3.18 앐 0.20 3.63 앐 0.18 3.31 앐 0.19
Total HDL 1.50 앐 0.10 1.54 앐 0.08 1.44 앐 0.07 1.55 앐 0.08
2
HDL
2
0.23 앐 0.03 0.25 앐 0.03 0.23 앐 0.04 0.34 앐 0.05
2
HDL
3
1.26 앐 0.08 1.29 앐 0.07 1.21 앐 0.05 1.21 앐 0.05
HDL
2
:HDL
3
0.20 앐 0.03 0.21 앐 0.03 0.19 앐 0.03 0.28 앐 0.05
LDL:HDL 2.15 앐 0.17 2.13 앐 0.16 2.63 앐 0.21 2.26 앐 0.21
3
Plasma triacylglycerol (mmol/L) 1.60 앐 0.16 1.51 앐 0.18 1.62 앐 0.12 1.64 앐 0.13
VLDL triacylglycerol (mmol/L) 0.66 앐 0.08 0.56 앐 0.07 0.76 앐 0.09 0.78 앐 0.7
VLDL cholesterol (mmol/L) 0.28 앐 0.05 0.24 앐 0.03 0.30 앐 0.04 0.33 앐 0.04
VLDL apolipoprotein B (
g/mL) 43.91 앐 6.36 39.93 앐 5.0 42.03 앐 3.57 49.09 앐 4.64
4
1
All values are x 앐 SEM. VLDL, very-low-density lipoprotein.
2,3
Significant difference within group after 8-wk supplementation (three-way ANOVA):
2
P 쏝 0.05,
3
P 쏝 0.01.
4
Significant difference between groups after 8 wk of supplementation, P 쏝 0.05 (analysis of covariance).
CLA AND TYPE 2 DIABETES MELLITUS 893
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more easily oxidized because of its structure, and this would
allow it to bypass a number of rate-limiting steps in the peroxi-
somal

-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 elon-
gation pathways. Consequently, t10,c12 CLA is not efficiently
incorporated into plasma lipids.
In conclusion, CLA supplementation did not have a positive
effecton insulin andglucose concentrations in persons with type
2 diabetes. Whereas we did show positive effects of CLA on
HDL-cholesterol and fibrinogen concentrations, the relative im-
portance of one CVD risk factor over another is unknown. Com-
pliancewasensured,giventhesignificantincreaseinc9,t11CLA
in total plasma lipid fraction, which was comparable with pre-
viously reported data (17, 52). Any truly effective dietary strat-
egywouldimproveCVD risk factorswithoutany negative effect
on other components of the metabolic syndrome.
We are indebted to the study participants for their enthusiasm and com-
mitmenttothestudyprotocolandtoKimJacksonforheraidinthelipoprotein
analysis.
FMprepared themanuscriptand conductedmostoftheexperiments. TPY
conducted the clinicalinvestigations.AMassistedin the experimental work.
JJNwas involvedin theformulation ofthescientifichypothesisforthisstudy
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 noneof the authors hadany other
conflict of interest.
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