AJCN. First published ahead of print August 10, 2016 as doi: 10.3945/ajcn.116.137646.

Distinct lipid profiles predict improved glycemic control in obese,

nondiabetic patients after a low-caloric diet intervention: the Diet,
Obesity and Genes randomized trial1,2
Armand Valsesia,3* Wim HM Saris,4 Arne Astrup,5 J
org Hager,3 and Mojgan Masoodi3,6*
3
Nestle Institute of Health Sciences, Lausanne, Switzerland; 4Department of Human Biology, School of Nutrition and Translational Research in Metabolism,
Maastricht University Medical Centre+, Maastricht, Netherlands; 5Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Copenhagen, Denmark; and 6Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Canada

ABSTRACT
Background: An aim of weight loss is to reduce the risk of type 2
diabetes (T2D) in obese subjects. However, the relation with longterm glycemic improvement remains unknown.
Objective: We evaluated the changes in lipid composition during
weight loss and their association with long-term glycemic improvement.
Design: We investigated the plasma lipidome of 383 obese, nondiabetic patients within a randomized, controlled dietary intervention
in 8 European countries at baseline, after an 8-wk low-caloric diet
(LCD) (8001000 kcal/d), and after 6 mo of weight maintenance.
Results: After weight loss, a lipid signature identified 2 groups of
patients who were comparable at baseline but who differed in their
capacities to lose weight and improve glycemic control. Six months
after the LCD, one group had significant glycemic improvement
[homeostasis model assessment of insulin resistance (HOMA-IR)
mean change: 20.92; 95% CI: 21.17, 20.67)]. The other group
showed no improvement in glycemic control (HOMA-IR mean
change: 20.26; 95% CI: 20.64, +0.13). These differences were sustained for $1 y after the LCD. The same conclusions were obtained
with other endpoints (Matsuda index and fasting insulin and glucose
concentrations). Significant differences between the 2 groups were
shown in leptin gene expression in adipose tissue biopsies. Significant
differences were also observed in weight-related endpoints (body
mass index, weight, and fat mass). The lipid signature allowed prediction of which subjects would be considered to be insulin resistant
after 6 mo of weight maintenance [validations receiver operating
characteristic (ROC) area under the curve (AUC): 71%; 95% CI:
62%, 81%]. This model outperformed a clinical dataonly model
(validations ROC AUC: 61%; 95% CI: 50%, 71%; P = 0.01).
Conclusions: In this study, we report a lipid signature of LCD
success (for weight and glycemic outcome) in obese, nondiabetic
patients. Lipid changes during an 8-wk LCD allowed us to predict
insulin-resistant patients after 6 mo of weight maintenance. The determination of the lipid composition during an LCD enables the identification
of nonresponders and may help clinicians manage metabolic outcomes
with further intervention, thereby improving the long-term outcome and
preventing T2D. This trial was registered at clinicaltrials.gov as
NCT00390637.
Am J Clin Nutr doi: 10.3945/ajcn.116.137646.
Keywords: adipose tissue, dietary intervention, insulin resistance,
lipidemia, obesity, weight loss

INTRODUCTION

Obesity is a major risk factor for a number of comorbidities

including cardiovascular disease, dyslipidemia, hypertension, insulin resistance, type 2 diabetes (T2D),7 and cancer (13). Excess
fat mass is associated with insulin resistance, but the underlying
mechanisms are not fully understood.
Caloric restriction reduces fat mass and also affects adipose
tissue function. Adipose tissue is important in energy homeostasis
and responds dynamically to caloric intake (46) by changing
the lipolysis rate and adipose tissue triacylglycerol turnover. Both
glyceroneogenesis and de novo lipogenesis respond to dietary energy
intake.
Multiple studies have shown that weight loss through energyrestricted dietary interventions improved dysfunctions involved
in the metabolic syndrome (7, 8). Nevertheless, a large interindividual variability was observed regarding the capacity to lose and
maintain weight (9). In clinical practice, a dietary intervention is
done with the use of a caloric-restricted diet and is followed by
a weight-maintenance (stabilization) phase. However, the long-term
outcomes of these dietary interventions are not predictable because
of the lack of knowledge including the complexity of adipose tissue
adaptation that results from the interventions. Although initial BMI
has some predictive value for intervention success (10), patients with
1

Supported by the European Commission, Food Quality and Safety

Priority of the Sixth Framework Program (FP6-2005-513946) and the Nestle
Institute of Health Sciences. This is a free access article, distributed under
terms (http://www.nutrition.org/publications/guidelines-and-policies/license/) that
permit unrestricted noncommercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.
2
The funders had no role in the study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
*To whom correspondence should be addressed. E-mail: armand.valsesia@
rd.nestle.com (A Valsesia), mojgan.masoodi@rd.nestle.com (M Masoodi).
7
Abbreviations used: CID, clinical intervention day; Diogenes, Diet, Obesity and Genes; FDR, false discovery rate; LCD, low-calorie diet; mRNA,
messenger RNA; PC, principal component; PCA, principal component analysis; PC1, first principal component; ROC, receiver operating characteristic;
T2D, type 2 diabetes.
Received May 11, 2016. Accepted for publication June 27, 2016.
doi: 10.3945/ajcn.116.137646.

Am J Clin Nutr doi: 10.3945/ajcn.116.137646. Printed in USA. 2016 American Society for Nutrition

Copyright (C) 2016 by the American Society for Nutrition

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2 of 10

VALSESIA ET AL.

similar starting BMIs have different responses. The rate of weight

loss alone is also a poor predictor and monitor for sustained weight
maintenance (11). More-objective molecular biomarkers are scarce
(12). In addition, the long-term relation between weight loss, the
improvement of lipid profiles, and glycemic control remains to be
explored.
In this report, we investigated changes in lipidemia in overweight, nondiabetic subjects from the Diogenes (Diet, Obesity
and Genes) study (clinicaltrials.gov; NCT00390637), which is
one of the largest dietary intervention studies. We studied how
changes in lipid profiles after a low-caloric diet would be associated with long-term improvements (after 6 mo of weight
maintenance) in both weight and glycemic control. We also
investigated whether a comprehensive lipidomic approach would
enable the prediction of insulin-resistant subjects despite the
subjects having completed a weight-loss and weight-maintenance
intervention and whether such a model would perform signifi-

cantly better than a model that was based only on easily accessible clinical variables.

METHODS

Study design
The Diogenes study was a multicenter, randomized, controlled
dietary intervention study that involved 8 European countries (13,
14) . A flowchart that describes the intervention and the sample size
at each phase is shown in Figure 1. Briefly, 938 overweight or obese,
nondiabetic adults [BMI (in kg/m2) between 27 and 45 and blood
fasting glucose concentrations ,6.1 mmol/L] underwent an 8-wk
weight-loss phase with the use of a complete meal-replacement
low-calorie diet (LCD). The LCD provided 800 kcal/d (Modifast;
Nutrition et Sante France). Participants could eat #400 g vegetables (corresponding to a maximal addition of 200 kcal/d).

FIGURE 1 Diogenes study. CID, clinical intervention day; Diogenes, Diet, Obesity and Genes; HP-HGI, high protein content and high glycemic index;
HP-LGI, high protein content and low glycemic index; LCD, low-calorie diet; LP-HGI, low protein content and high glycemic index; LP-LGI, low protein
content and low glycemic index; WMD, weight-maintenance diet.

LIPID PROFILES AND GLYCEMIC CONTROL AFTER LCD

Participants who achieved .8% weight loss (n = 773) were

randomly assigned to consume a weight-maintenance diet for
26 wk. The diets corresponded to a 2-by-2 factorial design as follows: diets with a high or low content of protein and a high or
low glycemic index or a control diet according to local food
customs (15). All diets had a moderate fat content (2530% of
total energy consumed) and were consumed ad libitum. Food
intake was monitored through food diaries (validated by a nutritionist). Of 773 participants, 548 subjects (71%) completed the
6-mo weight-maintenance intervention. Such a dropout rate
(29%) has been discussed in details previously (14) and was in
line with reports from other dietary studies (16, 17).
Two centers (Netherlands and Denmark) provided most of the
food to participants during the weight-maintenance intervention
(following recommendations from dietitians and in accordance
with the participants randomized diet). These 2 centers, which
were referred to as shop centers, extended the weight-maintenance
protocol up to 1 y. The remaining centers provided participants
with dietary instructions for the 6-mo period. Additional details
are presented in Moore et al. (15).
The analysis described here included 383 participants who
completed the entire intervention (LCD and $6 mo of weight
maintenance) and had available plasma samples. Plasma lipids
were profiled at baseline [clinical intervention day (CID) 1],
after 8-wk of LCD (CID2), and after 6 mo of weight maintenance (CID3).

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and CID2. Expression fold changes were scaled to a mean of

zero and unit variance before the PCA.
Clinical endpoints
The following clinical endpoints were included in the analysis:
BMI and weight; fat mass and fat-free mass were measured with
the use of dual-energy X-ray absorptiometry. The following glycemic control measures were analyzed: HOMA-IR, fasting glucose
and insulin, and the Matsuda index, which is a measure of insulin
sensitivity derived from 2-h oral glucose tolerance tests (21).
Total lipid concentrations (cholesterol, triglycerides, HDL, and
derived LDL determined with the use of Friedwalds formula)
were also analyzed with blood biochemistry.
Transcriptomics analysis
Abdominal subcutaneous adipose tissue biopsies were obtained by needle aspiration under local anesthesia after an
overnight fast at baseline, at the end of the LCD, and at the end
of weight maintenance. Biopsy samples were stored at 2808C
until total RNA extraction, and leptin messenger RNA (mRNA)
concentrations were assessed with the use of high-throughput
real-time polymerase chain reaction (22). Data are available from the Gene Expression Omnibus (series GSE60946,
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60946).
Analyses focused on 93 subjects who had available lipidomic and
transcriptomic data. The change in leptin expression was assessed
with the use of a 2-sided t test.

Ethics
Local ethics committees approved the study, written informed
consent was obtained from each patient, and the study was carried out in accordance with the principles of the Declaration of
Helsinki.

Lipidomic analysis
Liquid chromatographymass spectrometry data generation
has been described previously (18). A mixture of internal standards and calibration standards was added to each sample and
was followed by liquidliquid extraction with a (2:1) mixture of
dichloromethane and methanol according to the procedure described in detail in Hu et al. (19). The organic phase that contained
most of the lipids was removed and prepared for further analysis.
Extracted lipids were separated on a Ascentis Express C8 2.1 9
150-mm (2.7-mm particle size) column (Sigma-Aldrich) with the
use of an Acquity UPLC system (Waters) and analyzed with the
use of quadrupole time-of-flight mass spectrometry (Agilent Technologies). Experimental conditions during the chromatographic
analysis and detection by mass spectrometry were similar to the
protocol described by Hu et al. (19). In total, 125 intact lipids
including triacylglycerides, phosphocholines sphingomyelins,
cholesterol esters, cholesterols, and diacylglycerides were
measured.
A differential expression analysis was performed with the use
of paired t test (2 sided with unequal variance). P values were
adjusted for multiple testing with the use of the BenjaminiHochberg procedure (20). Differentially expressed lipids were
combined into a signature by performing a principal component
analysis (PCA) on their expression fold changes between CID1

Statistical analysis
Changes in clinical endpoints were tested with the use of linear
mixed-effect models. The lipid signature, sex, and age were
modeled as fixed effects; the center was modeled as a random
effect. P values were adjusted for multiple testing (20).
Prediction analyses were aimed at predicting subjects who
were classified as insulin resistant after weight maintenance (i.e.,
at CID3) with the use of data that were available either at CID1 or
during the LCD (CID1 and CID2). To ensure the robustness of the
models, the following definitions of insulin resistance at CID3
were used: Matsuda index ,3 and ,2.5 (21) and HOMA-IR $3
and $2.5 (23). The Matsuda index was derived from oral-glucosetolerance test (21), whereas the HOMA-IR (23) was derived from
fasting concentrations of glucose and insulin. Both variables have
been shown to correlate well with euglycemic insulin clamps and
to adapt for the assessment of insulin-resistance in nondiabetic
subjects (21, 2426). For each insulin-resistance definition, the
following 2 clinical models were constructed: one model used
baseline characteristics, and the other model used the percentage
of change during the LCD for the same clinical variables. None
of these models used variables at CID3. Clinical models included
the following variables: age, sex, BMI, lipid concentrations from
blood biochemistry (total triglyceride, total cholesterol, derived
LDL, and HDL), and either the Matsuda index or HOMA-IR
(according to the insulin-resistance definition that had been used).
A third combined clinical and lipidomic model was created by
adding the lipid signature during the LCD to the most-predictive
clinical model. Models were trained on the shop centers (Denmark
and Netherlands), and the predictive performance was evaluated
on the remaining centers (independent validation set). Such

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VALSESIA ET AL.

a setting provided an independent replication of the predictive

models. Only the prediction performance on the validation set is
reported; the performance on the training set is, by definition,
significantly higher and should not be used for evaluating the
utility of a model. Models were fitted with the use of logistic
regression and receiver operating characteristic (ROC) curves, and
a model evaluation was performed with the use of the pROC
package (27). A statistical comparison between ROC curves was
performed with the use of Delongs test (28). The specificity and
sensitivity of a model were determined with the use of the Youden
index (29). All analyses were performed with the use of R software (v3.0.0, www.r-project.org).
RESULTS

Baseline characteristics and overall clinical outcome

Patients baseline characteristics are described in Table 1.
The study included 383 of 548 patients (70%) who completed
the Diogenes intervention (14) (Figure 1). Patient inclusion for
lipidomic analyses was defined by the availability of plasma
samples at the 3 CIDs. Patients included in the lipidomic analyses were representative of the full cohort, and no sampling bias
was detected (for both baseline characteristics and the change in
clinical outcomes).
Mean changes after the 2 interventions (LCD and weight
maintenance) are also shown in Table 1. After the LCD, significant
improvements occurred both in weight and glycemic control. After
6 mo of weight maintenance, weight changed by 210.59 kg (95%
CI: 211.28, 29.91 kg), the Matsuda index changed by +1.68
(95% CI: 1.33, 2.02), and the HOMA-IR changed by 20.57 (95%
CI: 20.81, 20.33). Pearsons correlation between changes in
BMI and the Matsuda index was 20.17 (95% CI: 20.27, 20.07).
This coefficient suggested rather modest improvements in both
weight loss and glycemic control. In addition, a significant interindividual variability was observed (BMI: CV: 63.28%; Matsuda
index CV: 187.93%). Finally, patients with moderate weight loss
were able to exhibit strong glycemic control improvements. These
observations prompted the need for stratification analyses to

distinguish between subjects who had strong weight and glycemic

improvements from those who had lesser improvements.
Analyses of differentially expressed lipids and derivation of
single lipid signature
One hundred twenty-five lipid species were identified in
plasma of 383 patients at all CIDs. Seventy-nine of the lipids
were shown to be differentially expressed. Figure 2A shows the
correlation between these lipids and displays the following 2
clear blocks of species: one block was mostly composed of
triacylglycerols, and the other block was mostly composed of
phosphatidylcholines and sphingomyelins.
We summarized the log 2-fold-changes during the LCD of the
79 identified lipids with the use of a PCA. The first principal
component (PC1) explained 33.8% of the variance, whereas other
components explained ,15% of the variance. Figure 2B shows
that triacylglycerols were the lipids with the strongest influence
on the PC1.
Associations between lipid signature and clinical outcomes
after LCD and weight maintenance
With the use of the previously mentioned lipid signature
(PC1), which corresponded to the changes in lipid profiles during
the LCD, we tested whether the signature would be associated
with weight and glycemic improvements after the 8-wk LCD.
And indeed, the signature was significantly associated with BMI,
weight, and all glycemic variables [false discovery rates (FDRs)
,5%]. A marginal association was observed with fat mass
(FDR: 5.3%). The statistical analysis with the use of the first 5
principal components (PCs) of the PCA showed that only PC1
was significantly associated with clinical outcomes (data not
shown).
We assessed whether this LCD lipid signature would be
associated with long-term outcomes (after weight maintenance). The lipid signature was significantly associated with
changes in weight-related outcomes (except for the change in
fat-free mass) and glycemic control after 6 mo of weight

TABLE 1
Clinical characteristics of the study population1
Variable
Age, y
BMI, kg/m2
Weight, kg
Fat mass, kg
Fat-free mass, kg
Matsuda index
HOMA-IR, SI units
Fasting glucose, mmol/L
Fasting insulin, mIU/mL
Fasting total cholesterol, mmol/L
Triglyceride, mmol/L
Fasting LDL, mmol/L
Fasting HDL, mmol/L

Baseline
42
34.30
100.22
40.19
59.89
5.42
2.85
5.04
10.74
4.84
1.38
3.00
1.21

6
6
6
6
6
6
6
6
6
6
6
6
6

6
4.91
17.67
11.44
12.95
3.25
1.78
0.60
6.36
1.02
0.65
0.89
0.34

Changes after LCD

relative to baseline

23.84 6
211.24 6
28.31 6
22.96 6
2.37 6
21.01 6
20.26 6
23.44 6
20.69 6
20.32 6
20.49 6
20.06 6

1.07
3.40
4.67
4.30
3.48
1.93
0.52
6.23
0.76
0.58
0.64
0.23

Changes after weight maintenance

relative to baseline

23.63 6
210.59 6
28.49 6
22.17 6
1.68 6
20.57 6
20.11 6
22.00 6
0.07 6
20.14 6
20.03 6
0.15 6

2.30
6.73
6.39
3.53
3.15
2.21
0.57
7.42
0.81
0.55
0.66
0.24

All values are means 6 SDs. Clinical characteristics are shown for the 383 subjects at baseline (clinical intervention
day 1) as well as changes after the LCD (clinical intervention day 2 2 clinical intervention day 1) and changes after weight
maintenance (clinical intervention day 3 2 clinical intervention day 1). LCD, low-calorie diet.
1

LIPID PROFILES AND GLYCEMIC CONTROL AFTER LCD

5 of 10

FIGURE 2 Differentially expressed lipids. (A) Correlation between fold changes during the LCD for the 79 differentially expressed lipids. (B)
Importance of lipids in the signature (the percentage of explained variance corresponds to the percentage of the sum of absolute loadings of
the principal component). Chol, cholesterol; DG, diacylglyceride; LCD, low-calorie diet; PC, phosphocholines; PE, phosphoethanolamine; SM,
sphingomyelin; TG, triacylglycerides.

maintenance. These results suggest that changes in lipid composition during an LCD may predict clinical changes after weight
maintenance.

(Table 2); therefore, differences observed during the LCD and after
weight maintenance could not be imputed to initial differences.
Changes in leptin gene expression from adipose tissue
support stratification

Patient stratification on the basis of lipidomic signature

To enable a visualization of the previously detailed results, we
compared an arbitrary dichotomization of the signature (into positive and negative values) with a clustering approach (k-means
clustering). These 2 approaches led to very similar clustering. Thus,
we kept the simple dichotomization, which allowed us to define 2
groups of subjects and facilitated the visualization of their clinical
differences.
The 2 groups had very distinct evolutions of their weight and
glycemic variables over the intervention (see mean changes per
group in Table 2). Figure 3 shows that the group with negative
coefficients (PC ,0, depicted by solid lines in Figure 3) had
consistent and significantly fewer weight and glycemic improvements than were shown in the other group (PC $0, depicted by
dashed lines in Figure 3). Specifically, patients from the PC ,0
group showed no significant improvement in glycemic control;
after 6 mo of weight maintenance, their mean HOMA-IR change
was 20.26 (95% CI: 20.64, 0.13). Conversely, patients from the
PC $0 group showed a significant improvement in glycemic
control (mean HOMA-IR changes: 20.92; 95% CI: 21.17,
20.67). These observations were supported with results from
other glycemic endpoints (Table 2).
For simplicity, in the rest of the article, we refer to the PC ,0
group as nonresponders and to the PC $0 group as responders. At
baseline, the 2 groups had comparable weight and glycemic control

To test whether the observed stratification would be consistent

with well-studied weight-loss biomarkers, we assessed leptin gene
expression in adipose tissue biopsies from a subset of subjects (n =
93). Figure 3 shows the clear difference in leptin expression over
the full study. After weight maintenance, we observed a significant
downregulation of leptin expression in responders (P = 1.98 3
1027). By contrast, nonresponders did not show significant
changes (P = 0.78). Differences between the 2 groups were significant (P = 0.0016). These findings were consistent with observed changes in weight and body composition. At baseline
(CID1), no significant difference was shown between the 2 groups
(P = 0.42), which indicated that there were no differences in leptin
production or sensitivity.
Association with long-term outcomes (1 y after LCD)
Two centers (Denmark and Netherlands) extended the weightmaintenance diet for an additional 26 wk, which provided
a fourth set of clinical data. The number of retained patients was
54 of 75 (72%) for Denmark and 47 of 74 (64%) for the
Netherlands. A significant association (FDR ,5%) was observed
between the LCD lipid signature and changes in weight and
glycemic outcomes 1 y after the LCD. These observations
provided additional support to differences observed after 6 mo
of weight maintenance.

6 of 10

1.12
3.61
3.79
3.31
3.89
1.55
0.55
5.10
0.77
0.60
0.68
0.24
6
6
6
6
6
6
6
6
6
6
6
6
24.08
212.18
28.53
23.63
3.29
21.48
20.38
24.92
20.94
20.62
20.64
20.02
0.99
2.99
5.27
4.89
2.88
2.13
0.46
6.80
0.69
0.41
0.58
0.22
6
6
6
6
6
6
6
6
6
6
6
6
23.63
210.42
28.14
22.44
1.59
20.60
20.15
22.19
20.47
20.06
20.35
20.10
0.7400
0.8581
0.4220
0.5930
0.3782
0.1233
0.1554
0.2424
0.0132
3.41 3 10210***
0.1719
0.0533
4.49
16.78
10.92
13.19
3.20
1.73
0.61
5.86
1.07
0.70
0.92
0.34
6
6
6
6
6
6
6
6
6
6
6
6
5.18
18.44
11.70
12.64
3.29
1.81
0.60
6.78
0.98
0.55
0.85
0.33

33.64
100.31
38.51
61.45
5.17
2.96
5.09
11.04
4.94
1.58
3.07
1.15
6
6
6
6
6
6
6
6
6
6
6
6
34.87
100.14
41.58
58.60
5.63
2.75
5.00
10.48
4.75
1.22
2.94
1.26
BMI, kg/m
Weight, kg
Fat mass, kg
Fat-free mass, kg
Matsuda index
HOMA-IR, SI units
Fasting glucose, mmol/L
Fasting insulin, mIU/mL
Fasting total cholesterol, mmol/L
Triglyceride, mmol/L
Fasting LDL, mmol/L
Fasting HDL, mmol/L

1
All values are means 6 SDs. Clinical characteristics for the 2 patient subgroups (nonresponders: n = 205; responders: n = 178) at baseline (clinical intervention day 1) as well as changes after the LCD (clinical
intervention day 2 2 clinical intervention day 1) and changes after weight maintenance (clinical intervention day 3 2 clinical intervention day 1). P values assessed the significance of the lipid signature on a given
clinical variable. *,**,***,xSignificance of P values after correction for multiple-testing: *FDR , 0.05, **FDR , 0.01, ***FDR , 0.001, xFDR , 0.10. FDR, false discovery rate; LCD, low-calorie diet.

6.52 3 10205***
1.04 3 10204***
3.31 3 10203**
0.3952
0.0097*
0.0034**
0.095
0.0014**
0.0766
8.09 3 10209***
0.4956
0.0027**
2.36
6.87
7.15
3.75
3.29
1.59
0.58
5.24
0.79
0.57
0.66
0.23
6
6
6
6
6
6
6
6
6
6
6
6
23.75
211.09
28.95
22.18
2.27
20.92
20.14
23.22
0.02
20.32
20.04
0.20
2.25
6.58
5.64
3.33
2.93
2.60
0.55
8.78
0.82
0.47
0.67
0.24
6
6
6
6
6
6
6
6
6
6
6
6
23.53
210.15
28.08
22.16
1.16
20.26
20.07
20.91
0.11
0.02
20.02
0.12
9.97 3 10 ***
1.12 3 10210***
0.0486x
0.1224
6.13 3 10204***
7.90 3 10206***
0.0001***
6.70 3 10206***
5.56 3 10212***
8.48 3 10239***
1.56 3 10205***
0.0199*

Responders
Nonresponders
Responders
Responders
Nonresponders

Variable

TABLE 2
Clinical characteristics of the 2 patient subgroups1

Baseline

Nonresponders

Changes after LCD

relative to baseline

211

Changes after weight maintenance

relative to baseline

VALSESIA ET AL.

Association with change in total lipid concentrations

We tested whether the LCD lipidomic signature was associated
with routine biochemistry tests of total HDL, triglycerides, cholesterol, and LDL. As expected, changes in total lipid concentrations during the LCD were correlated strongly with the LCD
lipidomic signature (Table 2). After 6 mo of weight maintenance,
only changes in total HDL and triglycerides remained associated
with the signature (FDRs ,5%) (Table 2); responders had a higher
increase in HDL and a stronger reduction of triglycerides.
At baseline, the 2 groups significantly differed in total triglycerides (FDR ,5%) (Table 2) with responders having significantly higher concentrations than nonresponders. Therefore,
the stronger triglyceride reduction in responders after the LCD
was not surprising. However, Figure 3 shows clear differences in
triglyceride profiles between responders and nonresponders.
After the LCD, nonresponders had marginal improvements
(mean triglyceride change: 20.06 mmol/L; 95% CI: 20.11,
0.00 mmol/L; P = 0.054). However, these small improvements
did not last. After weight maintenance, triglyceride concentrations for nonresponders were comparable to their initial baseline
concentrations.
Statistical analyses of total triglyceride and cholesterol baseline
concentrations showed that these variables were not associated
with weight or glycemic outcomes (neither after the LCD or after
weight maintenance). Joint analysis of these variables with the
lipid signature excluded total lipid concentrations as potential
confounding factors of the signature. Therefore, despite some
baseline differences between responders and nonresponders in total
lipid concentrations, the differences were not sufficient to predict
clinical outcomes and could not be used as a substitute of a morecomprehensive lipid characterization.

LCD lipid signature and prediction of insulin-resistant

patients after weight maintenance
These analyses were aimed at predicting which subjects would be
considered insulin resistant despite the weight-loss and -maintenance
interventions. Predictions were first performed with models that
were based on clinical biochemistry variables (i.e., without the
need to perform comprehensive lipidomic analyses). Two types of
clinical models were constructed. One model used information at
baseline (i.e., the CID1 clinical model), and the other model used
changes during the LCD (i.e., the LCD clinical model). Then,
a third model was evaluated and combined the best clinical model
with the lipid signature.
We first defined insulin-resistant patients as those with a
Matsuda index ,3 at CID3. In this setting, an LCD clinical
model was not significantly better than a random prediction [on
the validation set, the AUC was 51% (95% CI: 35%, 67%)]
(Figure 4A). Such a low performance was explained by the
nonsignificant correlations between the LCD clinical variables
and insulin-resistance outcome.
By contrast, the CID1 clinical model provided a marginally
better performance [AUC: 65% (95% CI: 53%, 77%), with
specificity and sensitivity equal to 50.90% and 75.34%, respectively]. The combined CID1 clinical and lipidomic model
provided a significantly better performance [AUC: 72% (95% CI:
61%, 83%),with specificity and sensitivity equal to 62.40% and
73.19%, respectively]. This combined model outperformed the

LIPID PROFILES AND GLYCEMIC CONTROL AFTER LCD

7 of 10

FIGURE 3 Mean (95% CI) differences between responders and nonresponders over weight-loss and weight-maintenance interventions. (AF) Changes in
BMI, the Matsuda index, total triglyceride concentrations, fat mass, HOMA-IR, and leptin gene expression, respectively. Each graph shows values at a given
CID for the 2 patient subgroups. Groups were defined only on the basis of the sign of their low-calorie diet lipid signature. Dashed lines correspond to subjects
with positive signature scores. Solid lines correspond to subjects with negative signature scores. Because of their clinical outcomes and for simplicity, patients
denoted by dashed lines (positive signature scores) are referred to as responders; other patients are referred to as nonresponders. For panels A-E, the sample
sizes were 205 nonresponders and 178 responders. For panel F, the sample sizes were 58 nonresponders and 35 responders. In all panels, P values were
obtained from an ANOVA that tested differences at a given CID between the 2 groups. ANOVAs were adjusted for sex, age, and participating center. A
significant time-by-group interaction was observed for changes in the Matsuda index, HOMA-IR, triglyceride, and leptin concentrations (false discovery rates
,5%). CID, clinical intervention day.

clinical model (Delongs P = 0.032), thereby showing the

clinical utility of the model that combined the clinical variables
with the lipid signature.
Analyses were repeated with the use of an insulin-resistance
definition that was based on the HOMA-IR (insulin resistance
defined as HOMA-IR $3). Results were comparable to those
obtained with a Matsuda-index definition (Figure 4B), whereby
the combined model had AUC: 71% (95% CI: 62%, 81%), with
specificity and sensitivity equal to 68.11% and 62.23%, respectively.
This combined model outperformed the best clinical model (P =
0.01). Note that, in this setting, the best clinical model was only
marginally better than random [AUC: 61% (95% CI: 50%,
71%), with specificity and sensitivity equal to 68.88% and
45.79%, respectively].
Analyses were also performed with the use of more-stringent
cutoffs (Matsuda index ,2.5 and HOMA-IR $2.5), and the
same conclusions were reached. This showed that our results
were not biased because of the choice of a glycemic variable or
its cutoff when defining insulin resistance.

DISCUSSION

In this study, we aimed to understand the relation between

altered lipid metabolism and long-term effects on glucose homeostasis. We investigated changes in lipidemia during weight loss
and weight maintenance and the association of specific lipids with
weight and glycemic control outcomes.
Differentially expressed lipids could be summarized into a signature, which stratified 2 response groups. One group (responders)
significantly reduced weight and improved glycemic control,
whereas the other group (nonresponders) showed significantly less
improvement in weight and no improvement in glycemic control.
Because all patients included in this analysis lost .8% of their
initial body weights after the LCD, the results challenge the paradigm that weight loss in obese patients is correlated with improved glycemic control and, hence, a reduction of T2D risk (30).
Differences in lipid profiles and their associations with metabolic outcomes were stable #1 y after the end of the LCD, which
suggested metabolic adaptation rather than short-term dietary
effects. Responders had a significant that a decrease in leptin
mRNA, whereas nonresponders showed no significant changes.

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VALSESIA ET AL.

FIGURE 4 Prediction of insulin-resistant subjects at CID3. (A) Prediction of subjects with a Matsuda index ,3 at CID. (B) Prediction of
subjects with a HOMA-IR $3 at CID3. ROC AUCs (95% CIs) are indicated. ROC curves for which the 95% CIs encompass 50% were not
significantly better than those shown by a random predictor (diagonal lines). Indicated P values were obtained from Delongs test and compared
the combined model (lipidomic and clinical) with the best clinical model. CID, clinical intervention day; LCD, low-calorie diet; PC1, first principal
component; ROC, receiver operating characteristic.

Leptin is downregulated after weight loss (22, 31, 32), which

corresponds to a long-term adaptation after an LCD (32). Leptin
also plays a role in improving insulin sensitivity in obese patients
(5, 31). Hence, the clinical characteristics of the patient groups
and their concentrations of leptin mRNA were consistent with
current knowledge about the clinical and metabolic impacts of
leptin.
Analyses of blood total lipids indicated differences in triglyceride metabolism between the 2 groups. Responders had
significantly higher baseline total triglyceride concentrations
than nonresponders did (1.58 compared with 1.22 mmol/L, respectively), but both groups were considered to be within the
normal range (,1.69 mmol/L). Although the 2 groups had
similar starting BMI and glycemia, responders likely had more
flexibility to improve metabolically because of the slightly
higher triglyceridemia, whereas nonresponders were already
close to the recommended optimal range of 1.13 mmol/L (33).
This result may raise the question of whether the current
threshold on hypertriglyceridemia should be revised to morestringent concentrations.
These observations from blood total lipids were consistent
with the lipidomic results. An investigation of the individual
triacylglycerol species revealed that responders showed a consistent decrease during the LCD that was maintained after weight
maintenance, as was expected (data not shown). By contrast,
nonresponders displayed a significantly lesser decrease for most
triacylglycerol species. However, a significant increase during the
LCD was observed for several triacylglycerols such as 55:2, 55:3,
52:2, 52:3, 54:3, 54:4, 56:6, and 57:2. Such an increase was only
seen during the LCD because CID3s concentrations were

equivalent to starting baseline (CID1) concentrations. These

results show the importance of studying triacylglycerol species
instead of total triglycerides.
In addition, total triglycerides and total cholesterol, as obtained
from classical blood biochemistry, could not explain differences
in clinical outcomes, which indicated that the comprehensive
elucidation of lipid species remains needed for prediction purpose. And indeed, a clinical model that was based on commonly
accessible variables, including total blood lipids, offered only
a moderate performance of the prediction of insulin-resistance
status after weight maintenance. By contrast, a combined lipidomic and clinical model outperformed the clinical model. On
a validation data set, the combined model increased the prediction performance by +10% (an increase of the ROC AUC from
61% to 71%), which represented strong improvements in both
specificity and sensitivity.
Our results are strengthened by the fact that the Diogenes
study is the largest weight-maintenance trial of its kind. It
combines extensive clinical data, long-term weight-maintenance
follow-up (both at 6 mo and 1 y), and gene expression from
adipose tissue biopsies. Our conclusions originated from the
consistency across multiple clinical endpoints, and results were
fully reproduced when splitting the cohort into distinct discovery
and validation sets. Our next study will investigate biological
mechanisms that underlie the identified stratification to moredeeply elucidate differences in triacylglycerol speciation.
In conclusion, our study shows the importance of the stratification of patient populations after an LCD intervention and
suggests that determining the lipid profile during weight loss
could provide a powerful tool to identify responders to an LCD.

LIPID PROFILES AND GLYCEMIC CONTROL AFTER LCD

The results challenge current practices of assessing total lipids

(cholesterol and triglycerides) rather than investigating lipid
composition. Mechanistic studies are needed to elucidate the
physiological and molecular differences between the 2 patient
groups. Finally, to our knowledge, our study is the first to link
changes in the lipid composition during weight-management
interventions in nondiabetic, obese patients with long-term
weight and glycemic outcomes as well as to propose new predictive models that outperform clinical models for the prediction of
insulin-resistant subjects. These findings may open new avenues in
clinical research to study mechanisms of insulin resistance in the
prevention of delay of T2D in obese patients, thereby providing
new tools to clinicians to better monitor patients in weight-loss
interventions.
We thank Hele`ne Ruffieux, Jerome Carayol, and Yariv Levy from the
Nestle Institute of Health Sciences for statistical discussions. We also thank
David Savage for fruitful scientific discussion and feedback; Jim Kaput
from the Nestle Institute of Health Sciences for useful feedback; and Nik
Papageorgiou for proofreading the manuscript.
The authors responsibilities were as followsAV, JH, and MM: designed
the lipidomic data analyses; AV and MM: performed the statistical analyses,
interpreted the results, wrote the manuscript, and had primary responsibility
for the final content of the manuscript; WHMS and AA: designed the Diogenes
study; and all authors: provided input into the writing of the manuscript and
read and approved the final manuscript. AV, JH, and MM are full-time employees at the Nestle Institute of Health Sciences SA. WHMS reported having
received research support from several food companies such as Nestle, DSM,
Unilever, Nutrition et Sante, and Danone as well as from pharmaceutical
companies such as GSK, Novartis, and Novo Nordisk. WHMS is a medical
consultant for N&S and is an unpaid scientific advisor for the International Life
Science Institute, ILSI Europe. AA reported having received grants and personal fees from Global Dairy Platform; personal fees from from McCain
Foods, McDonalds, Arena Pharmaceuticals Inc., Basic Research, Dutch
Beer Knowledge Institute, Gelesis, Novo Nordisk, Orexigen Therapeutics Inc.,
S-Biotek, Twinlab, and Vivus Inc.; grants from Arla Foods, the Danish
Dairy Research Council, Nordea Foundation (outside of the submitted
work); and royalties for the book first published in Danish as Verdens Bedste
Kur (Politiken, Copenhagen) and subsequently published in Dutch as Het beste
dieet ter wereld (Kosmos Uitgevers, Utrecht/Antwerpen), in Spanish as Plan
DIOGENES para el control del peso. La dieta personalizada inteligente
(Editorial Evergrcas, Leon), and in English as Worlds Best Diet (Penguin,
Australia).

REFERENCES
1. Haslam DW, James WPT. Obesity. Lancet 2005;366:1197209.
2. Dixon JB. The effect of obesity on health outcomes. Mol Cell Endocrinol 2010;316:1048.
3. Lean ME. Pathophysiology of obesity. Proc Nutr Soc 2000;59:
3316.
4. Franck N, Gummesson A, Jernas M, Glad C, Svensson P-A, Guillot G,
Rudemo M, Nystr
om FH, Carlsson LMS, Olsson B. Identification of
adipocyte genes regulated by caloric intake. J Clin Endocrinol Metab
2011;96:E4138.
5. Havel PJ. Update on adipocyte hormones regulation of energy balance and carbohydrate/lipid metabolism. Diabetes 2004;53:S143
51.
6. Yu Y-H, Ginsberg HN. Adipocyte signaling and lipid homeostasis
sequelae of insulin-resistant adipose tissue. Circ Res 2005;96:1042
52.
7. Soare A, Weiss EP, Pozzilli P. Benefits of caloric restriction for cardiometabolic health, including type-2 diabetes mellitus risk. Diabetes
Metab Res Rev 2014;30(Suppl 1):417.
8. Alves NEG, Enes BN, Martino HSD, Alfenas R CG, Ribeiro SMR.
Meal replacement based on Human Ration modulates metabolic risk
factors during body weight loss: a randomized controlled trial. Eur J
Nutr 2014;53:93950.

9 of 10

9. Neiberg RH, Wing RR, Bray GA, Reboussin DM, Rickman AD,
Johnson KC, Kitabchi AE, Faulconbridge LF, Kitzman DW, Espeland
MA, et al. Patterns of weight change associated with long-term weight
change and cardiovascular disease risk factors in the Look AHEAD
Study. Obesity (Silver Spring) 2012;20:204856.
10. Baltasar A, Perez N, Serra C, Bou R, Bengochea M, Borrs F. Weight
loss reporting: predicted body mass index after bariatric surgery.
Obes Surg 2011;21:36772.
11. Purcell K, Sumithran P, Prendergast LA, Bouniu CJ, Delbridge E,
Proietto J. The effect of rate of weight loss on long-term weight
management: a randomised controlled trial. Lancet Diabetes Endocrinol 2014;2:95462.
12. Polsky S, Ogden LG, MacLean PS, Giles ED, Brill C, Wyatt HR.
Biomarker profile does not predict weight loss success in successful
and unsuccessful diet-reduced obese individuals: a prospective study.
ISRN Obes 2013;2013:804129.
13. Larsen TM, Dalskov S, van Baak M, Jebb S, Kafatos A, Pfeiffer A,
Martinez JA, Handjieva-Darlenska T, Kunesov M, Holst C, et al. The
Diet, Obesity and Genes (Diogenes) Dietary Study in eight European
countries - a comprehensive design for long-term intervention. Obes
Rev 2010;11:7691.
14. Larsen TM, Dalskov S-M, van Baak M, Jebb SA, Papadaki A,
Pfeiffer AFH, Martinez JA, Handjieva-Darlenska T, Kunesov M,
Pihlsgard M, et al. Diets with high or low protein content and glycemic
index for weight-loss maintenance. N Engl J Med 2010;363:2102
13.
15. Moore CS, Lindroos AK, Kreutzer M, Larsen TM, Astrup A, Van Baak
MA, Handjieva-Darlenska T, Hlavaty P, Kafatos A, Kohl A, et al.
Dietary strategy to manipulate ad libitum macronutrient intake, and
glycaemic index, across eight European countries in the Diogenes Study.
Obes Rev 2010;11:6775.
16. Miller M, Beach V, Sorkin JD, Mangano C, Dobmeier C, Novacic D,
Rhyne J, Vogel RA. Comparative effects of three popular diets on
lipids, endothelial function, and C-reactive protein during weight
maintenance. J Am Diet Assoc 2009;109:7137.
17. Hession M, Rolland C, Kulkarni U, Wise A, Broom J. Systematic review of randomized controlled trials of low-carbohydrate vs. low-fat/
low-calorie diets in the management of obesity and its comorbidities.
Obes Rev 2009;10:3650.
18. Szymanska E, van Dorsten FA, Troost J, Paliukhovich I, van Velzen
EJJ, Hendriks MMWB, Trautwein EA, van Duynhoven JPM, Vreeken
RJ, Smilde AK. A lipidomic analysis approach to evaluate the response to cholesterol-lowering food intake. Metabolomics 2012;8:
894906.
19. Hu C, van Dommelen J, van der Heijden R, Spijksma G, Reijmers TH,
Wang M, Slee E, Lu X, Xu G, van der Greef J, et al. RPLC-ion-trapFTMS method for lipid profiling of plasma: method validation and
application to p53 mutant mouse model. J Proteome Res 2008;7:4982
91.
20. Benjamini Y, Hochberg Y. Controlling the false discovery rate:
a practical and powerful approach to multiple testing. J R Stat Soc B
1995;57:289300.
21. Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from
oral glucose tolerance testing: comparison with the euglycemic insulin
clamp. Diabetes Care 1999;22:146270.
22. Viguerie N, Montastier E, Maoret J-J, Roussel B, Combes M, Valle C,
Villa-Vialaneix N, Iacovoni JS, Martinez JA, Holst C, et al. Determinants of human adipose tissue gene expression: impact of diet,
sex, metabolic status, and cis genetic regulation. PLoS Genet 2012;8:
e1002959.
23. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF,
Turner RC. Homeostasis model assessment: insulin resistance and
b-cell function from fasting plasma glucose and insulin concentrations
in man. Diabetologia 1985;28:4129.
24. Emoto M, Nishizawa Y, Maekawa K, Hiura Y, Kanda H, Kawagishi T,
Shoji T, Okuno Y, Morii H. Homeostasis model assessment as a clinical
index of insulin resistance in type-2 diabetic patients treated with
sulfonylureas. Diabetes Care 1999;22:81822.
25. Bonora E, Targher G, Alberiche M, Bonadonna RC, Saggiani F, Zenere
MB, Monauni T, Muggeo M. Homeostasis model assessment closely
mirrors the glucose clamp technique in the assessment of insulin sensitivity: studies in subjects with various degrees of glucose tolerance
and insulin sensitivity. Diabetes Care 2000;23:5763.

10 of 10

VALSESIA ET AL.

26. Stumvoll M, Mitrakou A, Pimenta W, Jenssen T, Yki-Jarvinen H, Haeften

TV, Renn W, Gerich J. Use of the oral glucose tolerance test to assess
insulin release and insulin sensitivity. Diabetes Care 2000;23:295301.
27. Robin X, Turck N, Hainard A, Tiberti N, Lisacek F, Sanchez J-C,
Muller M. pROC: an open-source package for R and S+ to analyze and
compare ROC curves. BMC Bioinformatics 2011;12:77.
28. DeLong ER, DeLong DM, Clarke-Pearson DL. Comparing the areas
under two or more correlated receiver operating characteristic curves:
a nonparametric approach. Biometrics 1988;44:83745.
29. Youden WJ. Index for rating diagnostic tests. Cancer 1950;3:325.
30. Hamman RF, Wing RR, Edelstein SL, Lachin JM, Bray GA, Delahanty L,
Hoskin M, Kriska AM, Mayer-Davis EJ, Pi-Sunyer X, et al. Effect of
weight loss with lifestyle intervention on risk of diabetes. Diabetes Care
2006;29:21027.

31. Siklova-Vitkova M, Klimcakova E, Polak J, Kovacova Z, Tencerova

M, Rossmeislova L, Bajzova M, Langin D, Stich V. Adipose tissue
secretion and expression of adipocyte-produced and stromavascular
fraction-produced adipokines vary during multiple phases of weightreducing dietary intervention in obese women. J Clin Endocrinol
Metab 2012;97:E117681.
32. Sumithran P, Prendergast LA, Delbridge E, Purcell K, Shulkes A,
Kriketos A, Proietto J. Long-term persistence of hormonal adaptations
to weight loss. N Engl J Med 2011;365:1597604.
33. Miller M, Stone NJ, Ballantyne C, Bittner V, Criqui MH, Ginsberg HN,
Goldberg AC, Howard WJ, Jacobson MS, Kris-Etherton PM, et al.
Triglycerides and cardiovascular disease: a scientific statement from the
American Heart Association. Circulation 2011;123:2292333.