The "Metabolically-obese Normal-weight" Phenotype in Two Asian Ethnic Groups and Its Reversal by Calorie Restriction
Phase: NA
Conditions: Glucose Metabolism Disorders, Obesity, Visceral, Obesity; Endocrine, Insulin Sensitivity/Resistance
Sponsor: Singapore Institute of Food and Biotechnology Innovation

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The “metabolically-obese normal-weight” phenotype in two Asian ethnic groups and its reversal 
by calorie restriction and exercise 
 
SPECIFIC AIMS & HYPOTHESES 
 
The prevalence of overweight and obesity in Singapore is approximately half of that in the United States, 
yet the incidence of type 2 diabetes is similar, and is expected to double in the near future.  This indicates 
that metabolic dysfunction, particularly insulin resistance, is widely prevalent even among individuals who 
are considered normal-weight or lean by conventional measures, i.e. body mass index (BMI) and percent 
body fat.  These individuals are often referred to as “metabolically-obese normal-weight” (MONW), and 
have increased risk for cardiometabolic disease despite their normal BMI and total body fat values.  The 
prevalence of the MONW phenotype varies across populations and differs markedly among different 
ethnicities (e.g. greater in Indian than in Chinese).  However, our understanding of the complex 
interactions between ethnicity, body composition, and metabolic dysfunction and its reversal remains 
rudimentary.  Previous attempts to characterize the MONW phenotype are confounded by the small but 
significant differences in BMI or percent body fat between groups (even if all subjects were lean, within 
the “normal” range), with MONW subjects being always “fatter” than the corresponding control subjects.  
There are no published studies that prospectively recruited groups of metabolically healthy and unhealthy 
lean individuals matched on BMI and percent body fat.  Furthermore, although weight loss improves body 
composition and many of the cardiometabolic abnormalities in most obese patients, little is known about 
the possible therapeutic effects of calorie restriction in MONW subjects.  Finally, regular endurance 
exercise can improve metabolic function, but its effects in MONW subjects are not known. 
 
Accordingly, a better understanding of the MONW phenotype and the evaluation of therapeutic appro-
aches for its reversal will have important implications for public health.  By facilitating earlier identification 
of these subjects, who are more likely to go undiagnosed and thus less likely to be treated before 
clinically overt cardiometabolic disease develops, results from this study will allow for earlier and effective 
intervention.  To this end, this project aims to test the following hypotheses:  
 
Hypothesis 1:  We hypothesize that, compared with metabolically-healthy lean subjects, MONW subjects 
have reduced insulin-mediated glucose disposal, impaired β-cell function, increased intra-abdominal 
adipose tissue, more ectopic fat deposition in liver and muscle, and decreased cardio-respiratory fitness.  
We further hypothesize that abnormalities along the “muscle axis” (intra-myocellular lipid content, insulin-
mediated glucose disposal, and cardio-respiratory fitness) are more pronounced in MONW subjects of 
Indian descent, whereas abnormalities along the “visceral axis” (intra-hepatic fat content, intra-abdominal 
adipose tissue, and β-cell function) are more pronounced in MONW subjects of Chinese descent.   
 
Hypothesis 2:  We hypothesize that, compared with a control condition of weight maintenance (i.e. no 
change in diet and physical activity), diet-induced mild weight loss (5 % of initial body weight) reduces 
ectopic fat deposition and improves metabolic function in MONW subjects, whereas matched exercise-
induced weight loss causes greater, and exercise without weight loss causes smaller beneficial effects in 
ectopic fat deposition and metabolic function. 
 
To test these hypotheses, total body fat (dual-energy X-ray absorptiometry), intra-abdominal adipose 
tissue (magnetic resonance imaging), intra-myocellular and intra-hepatic lipid (magnetic resonance 
spectroscopy), insulin-mediated glucose disposal (hyperinsulinemic-euglycemic clamp), β-cell function 
(mixed meal tolerance test), and cardio-respiratory fitness (maximum oxygen consumption test) will be 
evaluated in metabolically healthy and unhealthy lean subjects of Indian and Chinese descents, matched 
for BMI and percent body fat (Hypothesis 1), and in MONW subjects before and after calorie restriction- 
or exercise- induced weight loss, exercise without weight loss, or weight maintenance (Hypothesis 2).   
 
Results from this project are expected to result in the efficient targeting of health promotion programs in 
Singapore and the development of effective lifestyle modification approaches to reduce cardiometabolic 
disease risk in metabolically-unhealthy lean individuals.  This will minimize healthcare expenditures, 
positively affect productivity, and ultimately improve the health and the quality of life of the Singaporean 
population, and inform public health programs in other countries. 

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BACKGROUND & CLINICAL SIGNIFICANCE 
 
Obesity and type 2 diabetes in Singapore 
The incidence of overweight and obesity has been increasing during the past 2–3 decades in Singapore, 
and is expected to increase further in the future [1].  By the year 2050, it is estimated that more than half 
of the population will be overweight or obese, defined as having a body mass index (BMI, calculated as 
the weight in kilograms divided by the square of height in meters) equal to or greater than 25 kg/m2 [1].  
This is likely responsible, at least in part, for the concomitant increase in obesity-related co-morbid condi-
tions, particularly type 2 diabetes [1, 2].  The relationship between BMI and the risk for type 2 diabetes in 
populations from the Asia-Pacific region is linear within a wide range of BMI values (from ~21 kg/m2 to 
~34 kg/m2), so that for every 2 kg/m2 increase in BMI (which corresponds to ~6 kg for a normal-weight 
person of average stature), the risk for developing type 2 diabetes rises by ~27 % [2].  In Singapore, the 
prevalence of type 2 diabetes is expected to double from 7.3 % in 1990 to ~15 % in 2050, predominantly 
as a result of the fattening of the population, with the burden being greater for those of Indian descent 
than those of Chinese descent [1].  This is expected to reduce productivity, inflate healthcare costs, and 
increase mortality among Singaporeans [1, 3].   
 
Metabolic dysfunction in normal-weight individuals  
The prevalence of type 2 diabetes in Singapore is similar to that in the Unites States, even though the 
prevalence of overweight and obesity (BMI ≥25 kg/m2) in Singapore is approximately half that in the US 
[4].  This observation corroborates findings from many studies showing that markers of metabolic 
dysfunction (e.g. hyperglycemia, hyperinsulinemia, insulin resistance, dyslipidemia, and hypertension) are 
highly prevalent among Singaporean adults even at normal BMI values, i.e. even among people who are 
considered “normal-weight” or “lean” by conventional measures [5, 6].  The existence of people who have 
normal body weight but also have metabolic dysfunction, and therefore greater risk for developing 
cardiometabolic disease, was recognized several decades ago [7, 8].  At the extreme of this paradigm, 
even among members of the Calorie Restriction Society who undergo self-imposed calorie restriction for 
years based on the belief that this will help them ensure a long and healthy life, there are many 
individuals (~40 %) with impaired glucose tolerance, despite very low BMI and total body fat [9].  These 
individuals are often referred to as “metabolically-obese normal-weight” (MONW) or “metabolically-
abnormal lean” or “metabolically-unhealthy lean” subjects.  The prevalence of this phenotype ranges from 
5 % to 45 % depending on the BMI and the metabolic criteria used for its definition, as well as the 
characteristics of the population (i.e. age, sex, and ethnicity) [10, 11].  Similar variability has been obser-
ved across Asia [12-16].  For example, among the Chinese, ~8 % of the population as a whole, or ~13 % 
of those who are considered lean by virtue of body fat percent (i.e. ≤25 % for men and ≤35 % for women), 
are metabolically unhealthy, defined as having three or more metabolic abnormalities characteristic of the 
metabolic syndrome [13].  Among Indians, on the other hand, 15–25 % of the population (or 20–40 % of 
those who are considered lean by virtue of BMI, i.e. <25 kg/m2) satisfies the criteria for metabolic 
syndrome [16, 17].  The MONW phenotype in Asians is associated with 3-fold greater risk for carotid 
atherosclerosis (i.e. cardiovascular disease) [14] and 4.5–8.5-fold greater risk for developing type 2 
diabetes [13].  In fact, MONW subjects have increased risk for cardiometabolic disease [13, 14] and 
greater all-cause mortality [18] not only compared to metabolically-healthy lean subjects, but also 
compared to metabolically-healthy obese subjects.  This underscores the importance of metabolic 
dysfunction independent of excess body weight and total adiposity. 
 
The MONW phenotype  
The mechanisms responsible for the development of metabolic abnormalities in lean people are not 
entirely clear.  The MONW phenotype can manifest early in life, e.g. during childhood [19], which 
corroborates the existence of genetic predisposition for metabolic dysfunction in the face of low BMI 
values [20].  Previous studies have identified a number of factors associated with the MONW phenotype, 
including increased intra-abdominal (visceral) adipose tissue, increased liver and muscle fat content, 
increased fat cell size, adipose tissue inflammation, altered inflammatory and adipokine profiles, reduced 
skeletal muscle mass, lack of physical activity, and low cardio-respiratory fitness [7, 9-11, 13, 16, 21-29].  
All of these factors have been directly or indirectly associated with insulin resistance (defined by a variety 
of methods), which is by far the commonest metabolic correlate of the MONW phenotype across all 
ethnicities, age groups, and sexes [7, 10, 24, 27].  In fact, the greater prevalence of the MONW pheno-
type in Indians [16, 17] than in the Chinese [13] mirrors results obtained recently by our team, showing 

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that among lean Singaporean men (BMI <25 kg/m2 or body fat ≤20 %), those of Indian descent have 
significantly lower insulin sensitivity, evaluated as the insulin-mediated glucose disposal rate during a 
hyperinsulinemic-euglycemic clamp procedure, compared to those of Chinese descent [30].  Similar 
results have been reported by other investigators in smaller groups of subjects [31] or when using simpler 
indices of insulin sensitivity [32, 33].  Therefore, an insulin resistant glucose metabolism, broadly defined 
by subnormal responses to physiological insulin concentrations [34], is the hallmark of the MONW 
phenotype. 
 
Owing to the lack of a consistent definition, there is some variability among studies in the phenotypic 
characterization of MONW subjects [11].  This is further complicated by the small but significant 
differences in BMI and, more commonly, percent body fat between groups of metabolically healthy and 
unhealthy lean subjects, with MONW subjects being always somewhat “fatter” (even though within the 
“lean” range) [7, 13, 16, 21-23, 28, 29].  Likewise, BMI and body fat are typically greater in relatively 
insulin-resistant (e.g. Indian) than in relatively insulin-sensitive (e.g. Chinese) individuals in studies 
reporting on ethnic differences in insulin action among lean people [30, 32].  This in itself could be 
responsible for the differences observed in metabolic function.  There is considerable (~2-fold range) 
variability between individuals in the percent body fat [35, 36] and the insulin-mediated glucose disposal 
rate (a direct measure of whole-body insulin sensitivity) [37] for the same BMI value within the normal-
weight range (i.e. BMI <25 kg/m2), so that people with the same BMI can have very different body fat and 
insulin sensitivity without this necessarily being associated with the presence or absence of generalized 
metabolic dysfunction.  Even among lean and metabolically-healthy Asians, total body fat is a major 
correlate of insulin-mediated glucose disposal [38].  It is thus possible that some of the reported 
differences between metabolically healthy and unhealthy lean subjects arise from normal variability and 
the differences in body fat between groups, rather than being an inherent characteristic of the MONW 
phenotype.  In support of this possibility, when metabolically healthy and unhealthy lean subjects (defined 
as those having normal and impaired glucose tolerance, respectively) were retrospectively matched on 
total body fat, there were no differences between phenotypes in circulating concentrations of metabolic 
and inflammatory markers (i.e. high-density lipoprotein (HDL)-cholesterol, triglycerides, free fatty acids, C-
reactive protein, adiponectin, and leptin) [9].  There are no studies that prospectively recruited groups of 
metabolically healthy and unhealthy lean individuals matched on BMI and percent body fat.  A deeper 
understanding of the MONW phenotype, as proposed here, is important to dissect the metabolic 
abnormalities that are inherent to the phenotype from those merely associated with differences in total 
body fat.  This will allow for proper identification and more efficient therapeutic targeting of MONW 
individuals, who are at greater risk for cardiometabolic disease.  
 
Weight loss and metabolic function  
Little is known about possible interventions for improving metabolic function in MONW subjects.  It is well 
established that diet-induced weight loss can improve body composition and many of the cardiometabolic 
abnormalities in most obese patients (e.g. decreases total body fat, intra-abdominal adipose tissue, and 
ectopic fat deposition in liver and muscle; increases insulin sensitivity; improves blood lipid profile; and 
reduces blood pressure) [39-48], so that a moderate 10 % weight loss has become the cornerstone of 
obesity treatment [49].  However, MONW individuals are by definition lean, so recommending even 
moderate amounts of weight loss may not be a feasible therapeutic target [50].  It is therefore important to 
better understand the metabolic effects of smaller amounts of weight loss.  Recently, we conducted a 
randomized controlled trial to evaluate the effects of mild weight loss (5 % of initial body weight) on 
cardiometabolic function in non-Asian subjects with obesity and insulin resistance, and found that even 
this small amount of weight loss decreases fat deposition in the liver and the intra-abdominal area, and 
increases insulin action in skeletal muscle, liver, and adipose tissue [51].  These results demonstrate that 
mild weight loss can improve many cardiometabolic abnormalities in metabolically-unhealthy obese 
subjects, but whether the same holds true for metabolically-unhealthy lean subjects is not known.  A 
small, non-randomized, single-arm study in 7 lean, insulin-resistant offspring of parents with type 2 
diabetes reported that modest ~6 % diet-induced weight loss reduced intra-myocellular lipid (i.e. fat within 
skeletal muscle fibers) content and increased insulin-mediated glucose disposal rate (both by ~30 % 
compared with baseline values), but did not significantly affect intra-abdominal adipose tissue volume or 
liver fat content [52].  It is thus not known whether mild diet-induced weight loss produces similar changes 
in body composition, fat distribution, and metabolic function in lean versus obese metabolically unhealthy 
subjects.  

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Endurance exercise and metabolic function  
Exercise is a very potent intervention that can readily improve metabolic function, particularly insulin 
sensitivity [53-55], and when performed under controlled conditions, can lead to weight loss and favorable 
changes in body composition [56-58].  For the same amount weight loss (8–9 % of initial body weight) 
induced by a low-calorie diet or endurance exercise, exercise causes a greater reduction in fat mass, 
minimizes the decrease in muscle mass, and increases cardio-respiratory fitness compared with calorie 
restriction [56].  Also, exercise-induced weight loss results in a greater reduction in intra-abdominal 
adipose tissue (by ~20 %) [56], a greater increase in insulin-mediated glucose disposal during a hyper-
insulinemic-euglycemic clamp (by ~30 %) [56], and a much greater reduction in the total insulin response 
to an oral glucose tolerance test (by ~2.5–fold) [57] compared with matched diet-induced weight loss, 
although these differences did not reach statistical significance.  These observations raise the possibility 
that exercise may be more advantageous than calorie restriction for improving metabolic function in 
MONW subjects.  This is an intriguing hypothesis given that the MONW phenotype is frequently 
associated with low levels of habitual physical activity, reduced muscle mass, and low cardio-respiratory 
fitness [9, 22, 25, 26, 28, 29].   
 
Knowledge gaps fulfilled by this project 
Our understanding of the complex interactions between ethnicity, body composition, and metabolic 
dysfunction and its reversal by calorie restriction and exercise remains rudimentary.  Accordingly, a better 
understanding of the MONW phenotype and the evaluation of the efficacy of different therapeutic 
approaches for its reversal will have important implications for public health, particularly in a multi-ethnic 
population, because it will help identify and intervene earlier in these subjects who are more likely to go 
undiagnosed and thus less likely to be treated before clinically overt cardiometabolic disease develops.  
To this end, this study aims to test the following hypotheses:  
 
Hypothesis 1:  We hypothesize that, compared with metabolically-healthy lean subjects, MONW subjects 
have reduced insulin-mediated glucose disposal, impaired β-cell function, increased intra-abdominal 
adipose tissue, more ectopic fat deposition in liver and muscle, and decreased cardio-respiratory fitness.  
We further hypothesize that abnormalities along the “muscle axis” (intra-myocellular lipid content, insulin-
mediated glucose disposal, and cardio-respiratory fitness) are more pronounced in MONW subjects of 
Indian descent, whereas abnormalities along the “visceral axis” (intra-hepatic fat content, intra-abdominal 
adipose tissue, and β-cell function) are more pronounced in MONW subjects of Chinese descent.   
 
Hypothesis 2:  We hypothesize that, compared with a control condition of weight maintenance (i.e. no 
change in diet and physical activity), diet-induced mild weight loss (5 % of initial body weight) reduces 
ectopic fat deposition and improves metabolic function in MONW subjects, whereas matched exercise-
induced weight loss causes greater, and exercise without weight loss causes smaller beneficial effects in 
ectopic fat deposition and metabolic function. 
 
METHODS / APPROACH 
 
Subjects 
A total of 120 men and women between 21 and 65 yrs old will participate in this study: 40 metabolically-
healthy lean subjects (20 Indian and 20 Chinese) and 80 metabolically-obese, normal-weight (MONW) 
subjects (40 Indian and 40 Chinese).  The two groups will be balanced on age, sex, ethnicity, BMI and 
percent body fat, to avoid confounding metabolic function outcomes due to differences in these factors.  
Matching for BMI and body fat is particularly important to circumvent the limitation in interpreting the 
results from previous studies in which MONW subject groups were “fatter” than the respective control 
groups of metabolically-healthy lean subjects [7, 21, 28].  As insulin resistance is the hallmark of the 
MONW phenotype [7, 10, 24, 27], we will use ethnicity- and sex- specific cutoff values [33] of the homeo-
stasis model assessment of insulin resistance (HOMA-IR) score, calculated from fasting plasma glucose 
and insulin concentrations [59], to classify subjects as metabolically healthy or MONW at screening.  
HOMA-IR scores >1.41 for Chinese men and women, and >2.17 and >1.99 for Indian men and women, 
respectively, will be considered indicative of metabolic dysfunction; these values correspond to the upper 
limit of the 95 % confidence interval of HOMA-IR (i.e. the most insulin resistant) in Singaporeans with 
normal glucose tolerance [33].   

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Based on the prevalence of the MONW phenotype in the Chinese and the Indians [13, 16, 17], who 
constitute ~85 % of the Singaporean population, we estimate we will need to screen ~650 individuals to 
identify 80 MONW and 40 metabolically-healthy subjects.  We do not anticipate any difficulties in 
screening and recruiting the required number of subjects for this study.  We intend to post flyers and 
disseminate information about the study to a large number of potential volunteers through our diabetes, 
lipid, and cardiometabolic clinics at the National University Health System Hospitals and general 
endocrinology clinics at the Tan Tock Seng Hospital.  This effort will be complemented by dissemination 
of information about the study through blast emails to students and employees at the National University 
of Singapore (NUS) and the Nanyang Technological University (NTU), posted flyers, and word-of-mouth.  
Written informed consent will be obtained from each subject before participation in the study.  During the 
screening visit at the CNRC, subjects will complete a comprehensive medical evaluation, including a 
history and physical examination, and standard blood tests.  They will also have their body fat percent 
measured by using dual-energy X-ray absorptiometry (DEXA).  Women of child-bearing potential will have 
a urine pregnancy test.  The following exclusion criteria will be used: i) BMI ≥25 kg/m2, but also <19 kg/m2 
(to avoid the risk of subjects becoming seriously underweight (i.e. BMI ≤18 kg/m2) after 5 % weight loss); 
ii) age <21 and >65 yrs; iii) use of medications that can affect metabolic function (including oral 
contraceptives and hormone replacement therapy); iv) regular use of tobacco products; v) regular 
consumption of alcohol; vi) pregnant or breastfeeding women; vii) evidence of significant organ system 
dysfunction or disease; viii) recent weight loss (≥5 % over the past 6 months); and ix) severe asthma and 
respiratory problems that prevent subjects from exercising.  
 
Experimental design 
An overview of the study flow is shown in Figure 1.  This project has a cross-sectional arm (observational 
study to evaluate differences between MONW and metabolically-healthy lean subjects from two different 
ethnic backgrounds; Hypothesis 1) and an intervention arm (randomized controlled trial to assess the 
effects of calorie restriction and exercise in MONW subjects; Hypothesis 2).  Inclusion of a metabolically-
healthy lean group is important for the unbiased characterization of the MONW phenotype (by comparing 
results for MONW subjects against a sex-, age-, ethnicity-, 
BMI- and percent body fat- matched group of metabolically-
healthy lean people), but also for the better evaluation of the 
effects of lifestyle intervention in MONW subjects (by 
comparing results after calorie restriction and exercise not only 
against values at baseline, but also against the “normal” range 
of values for lean people).  All outcomes will be assessed in 
exactly the same manner one time in metabolically-healthy 
lean subjects and two times in MONW subjects (once at 
baseline and again after the interventions), after making sure 
that participants have remained weight-stable for at least two 
weeks (≤1 % change in body weight/wk; both at baseline and 
after the interventions) to avoid potential confounding of our 
measures from acute changes in energy balance.  Following 
completion of baseline testing, MONW subjects will be 
randomly assigned to one of four intervention groups (n = 20 in 
each; 10 Indian and 10 Chinese): i) hypocaloric diet to induce 
5 % weight loss, ii) endurance exercise without changes in 
energy intake (i.e. isocaloric diet) to induce 5 % weight loss, iii) 
endurance exercise with a compensatory increase in energy 
intake to maintain body weight, or iv) no change in exercise 
and diet to maintain body weight (i.e. control). 
 
Following the successful screening and enrollment into the study, subjects will visit the CNRC and CIRC 
(both facilities are located in the same building at NUS campus) to have their resting metabolic rate, 
cardio-respiratory fitness, and body fat distribution assessed.  Two weeks later (diet lead-in period, during 
which the energy requirements for weight maintenance will be tailored for each subject), and provided 
body weight has remained stable (≤1 % change per week), subjects will visit the CNRC on two additional 
occasions, separated by 5–10 days, to undergo: i) a 3-hour hyper-insulinemic-euglycemic clamp 
procedure, to determine whole-body insulin-mediated glucose disposal and insulin sensitivity [30], and ii) 
Figure 1 Overview of study flow.
Fat distribution assessment
Mixed meal tolerance test
Diet-induced
5% weight loss
(n=20)
Metabolically-healthy
lean (n = 40)
20 Indian
20 Chinese
Medical screening and body composition
Metabolically-obese normal 
weight (MONW, n = 80)
40 Indian
40 Chinese
Hyperinsulinemic-euglycemic clamp
Exercise-induced
5% weight loss
(n=20)
Exercise with
no weight loss
(n=20)
Control
(n=20)
Body composition and fat distribution assessment
Mixed meal tolerance test
Hyperinsulinemic-euglycemic clamp
Metabolically-obese normal 
weight (MONW, n = 80)

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a 3-hour mixed meal tolerance test (MMTT), to determine glucose tolerance, insulin secretion, and β-cell 
function [60-62].  This battery of tests combines advanced medical imaging techniques to assess the 
amount of adipose tissue and its distribution in the body, with state-of-the-art (i.e. glucose uptake during 
infusion of insulin) as well as physiological (i.e. responses to meal ingestion) tests to assess metabolic 
function, thereby allowing for a deep and comprehensive phenotypic characterization of our subjects.  
 
Resting energy expenditure and cardio-respiratory fitness.  Subjects will visit the CNRC in the 
morning, after having fasted overnight, and having abstained from strenuous exercise, alcohol, and 
caffeine on the previous day.  Following 30 min of bed rest, they will have their oxygen consumption and 
carbon dioxide production measured continuously for 30 min, while they breathe under a ventilated hood, 
and resting metabolic rate (RMR) will be determined by using indirect calorimetry (Quark RMR; 
COSMED, Rome, Italy) [63].  Thereafter, they will undergo a graded exercise test (standard Bruce 
protocol [64, 65]) on the treadmill (Pro+; Cybex International, Medway, MA), while breathing through a 
face mask, to determine their maximal oxygen consumption (VO2max) (Metanalyzer 3B; CORTEX 
Biophysik GmbH, Leipzig, Germany).  This test involves increasing the treadmill speed and grade every 3 
minutes (to allow achievement of a steady state) until volitional exhaustion. 
 
Imaging for body composition.  Fat mass and fat-free mass will be determined by using dual-energy X-
ray absorptiometry (DEXA) on a Discovery QDR Series DEXA scanner (Hologic, Bedford, MA, USA) in 
the CNRC.  Intra-abdominal adipose tissue (visceral fat) and subcutaneous abdominal adipose tissue 
volumes will be determined by using magnetic resonance (MR) imaging.  Intra-hepatic triglyceride (IHTG) 
and intra-myocellular lipid (IMCL) contents will be determined by using proton MR spectroscopy.  MR 
imaging and spectroscopy will be conducted on a 3 Tesla MR scanner (Tim Trio, Siemens, Erlangen, 
Germany) in the CIRC; details of the scanning and analysis procedures have been described in detail 
previously [30].   
 
Metabolic testing 
For both metabolic testing visits (clamp and MMTT), which will be scheduled ~1 wk apart, subjects will 
come to the CNRC in the morning, after having fasted overnight.  They will be instructed to abstain from 
alcohol and caffeine consumption on the previous day, and from performing any strenuous exercise on 
the preceding 3 days (to avoid potential delayed metabolic effects of exercise).  Subjects will use food 
diaries given to them at screening to record all food and drinks consumed on the day before they come to 
the CNRC for their first metabolic testing visit, and will be instructed to replicate the same diet on the day 
preceding the remaining visits.  On all occasions, vital signs (temperature, heart rate, and blood pressure) 
will be obtained after 30 min of bed rest, and before any testing begins. 
 
Hyperinsulinemic-euglycemic clamp.  Whole-body insulin sensitivity will be determined by using a 3-
hour hyperinsulinemic-euglycemic clamp [30, 66].  Indwelling catheters will be inserted into an antecubital 
vein of one arm to infuse insulin and dextrose, and a forearm vein in the contralateral arm for blood 
sampling.  After a baseline fasting blood sample is obtained, insulin will be infused at a constant rate of 
50 mU/m2 body surface area/min for the duration of the clamp (180 min).  Plasma glucose concentration 
will be measured every 10 min at bedside and the infusion rate of 20 % dextrose solution will be adjusted 
accordingly to maintain euglycemia (100 mg/dL or 5.6 mmol/L).  At the beginning of this test, a muscle 
biopsy with be performed, which involves obtaining a small piece (the size of a corn kernel) of leg muscle 
(quadriceps) under sterile conditions and local anesthesia, to evaluate cellular factors involved in glucose 
handling by muscle. 
 
Mixed meal tolerance test (MMTT).  An indwelling catheter will be inserted into a forearm vein of one 
arm to obtain blood samples.  After a baseline fasting blood sample is obtained, a 600-kcal liquid mixed 
meal (55 % of energy from carbohydrate, 15 % from protein, and 30 % from fat) will be consumed over 5 
minutes (time will start counting at the beginning of the meal).  Blood samples to measure glucose, C-
peptide, and insulin concentrations will be collected according to the following schedule: 10, 20, 30, 45, 
60, 90, 120, 150, and 180 minutes.   
 
Diet and exercise interventions 

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Daily energy requirements for weight maintenance (i.e. isocaloric diet) will be estimated for each subject 
by multiplying resting energy expenditure (i.e. RMR) by a factor of 1.4 [67].  All MONW subjects will be 
prescribed an isocaloric diet for the 2 weeks preceding their first metabolic testing visit (diet lead-in 
period).  During this time, all food will be prepared in the metabolic kitchen of the CNRC and will be 
provided to all subjects, to familiarize them with food portions at the prescribed energy intake.  Body 
weight will be monitored and the energy content of the diet will be adjusted accordingly to maintain weight 
(≤1 % change per week).  This will ensure a fine-tuned, tailored determination of energy requirements for 
weight maintenance.  Thereafter, MONW subjects will be randomized to one of four intervention groups 
consisting of different manipulations of diet and exercise (Figure 2). 
 
Figure 2 Diet and exercise interventions and expected weight changes in MONW subjects randomized in different intervention groups.
Control
Diet-induced weight loss
Exercise-induced weight loss
Exercise without weight loss
Diet (energy intake)
↔
↓
↔
↑
Exercise (energy expenditure)
↔
↔
↑
↑
Expected weight change
↔
↓
↓
↔
 
 
MONW subjects randomized in the diet and exercise weight loss groups will participate in a supervised 
weight loss program to help ensure they are under a similar weekly energy deficit and achieve a 5 % 
weight loss at approximately the same time.  Participants in the diet-induced weight loss group (n = 20) 
will be prescribed a reduced-calorie diet (~500 kcal/d below their needs for weight maintenance), and will 
be instructed not to change their physical activity habits, in order to achieve a weekly weight loss of ~0.5 
kg.  Participants in the exercise-induced weight loss group (n = 20) will be prescribed an isocaloric diet 
and an endurance exercise program designed to elicit an energy deficit of ~500 kcal/d, to achieve the 
same weekly weight loss of ~0.5 kg.  Participants in the exercise without weight loss group (n = 20) will 
be prescribed the same endurance exercise program but their caloric intake will be increased accordingly 
to prevent any changes in body weight.  Finally, participants in the control group (n = 20) will be 
instructed to maintain their physical activity habits and consume an isocaloric diet to maintain their body 
weight.   
 
The macronutrient composition of the diet will be the same for all groups (55–60 % of energy from 
carbohydrate, 15–20 % from protein, and 20–30 % from fat); no vitamins or other nutritional supplements 
will be given.  The prescribed diet and exercise programs will be individualized based on each subject’s 
food and exercise preferences.  During the first week of the intervention, all food will be provided to 
subjects in the diet-induced weight loss group and the exercise without weight loss group, to ensure they 
get accustomed to the new prescribed energy intake (reduced and increased relative to their isocaloric 
needs, respectively).  Likewise, all exercise sessions for subjects in the exercise-induced weight loss 
group and the exercise without weight loss group will be supervised, to ensure they get familiarized with 
the exercise workload and the different options for exercise mode, intensity, and duration.  A member of 
the research team will be contacting the subjects on a weekly basis to monitor progress, foster adherence 
to diet and exercise prescription, identify possible problems, and build a supportive environment for the 
participant. 
 
Sample analyses 
Plasma glucose concentration will be determined by using the glucose oxidase method on an automated 
glucose analyzer (YSI 2300 Stat Plus; YSI Life Sciences, Yellow Spring, OH, USA).  Plasma insulin and 
C-peptide concentrations will be determined by using electrochemiluminescence technology (Roche/ 
Hitachi cobas e411 immunochemistry analyzer; Roche Diagnostics, Indianapolis, IN).  Total plasma trigly-
ceride and HDL-cholesterol concentrations will be determined by using a colorimetric and an immuno-
turbidimetric assay, respectively, on an automated analyzer (Roche/Hitachi cobas c311 clinical chemistry 
analyzer; Roche Diagnostics, Indianapolis, IN).  Plasma concentrations of leptin, adiponectin, C-reactive 
protein (CRP), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) will be measured by using 
commercially available enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems 
(Minneapolis, MN, USA), ALPCO Diagnostics (Salem, NH, USA), and EMD Millipore (Billerica, MA, USA). 
 
Calculations 

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Insulin sensitivity.  The average rate of dextrose infusion during the final 30 minutes of the clamp 
corresponds to the insulin-mediated whole-body glucose disposal (M value).  Insulin sensitivity will be 
calculated as the M/I ratio, i.e. glucose disposal divided by the steady-state plasma insulin concentration 
(i.e. average insulin concentration during the final 30 minutes of the clamp) [30].  The M/I ratio adjusts 
glucose disposal rate for potential differences in insulin concentrations attained during the clamp.   
β-Cell function.  The insulin secretion rate (ISR) response sensitivity to plasma glucose after ingestion of 
the mixed meal will be assessed by using oral minimal model analysis (SAAM II version 2) of plasma C-
peptide concentrations, to obtain Φ-static (ISR response sensitivity to plasma glucose concentration), Φ-
dynamic (ISR response sensitivity to the rate of change in plasma glucose concentration), and Φ-total 
(overall ISR response sensitivity to plasma glucose) [60-62].  The disposition index, which provides an 
assessment of the appropriateness of insulin secretion in relationship to peripheral insulin sensitivity, will 
be determined as the product of insulin sensitivity and insulin secretion [68, 69].   
Glucose tolerance.  Oral glucose tolerance will be determined by integrating (with the trapezoid rule) the 
area under the glucose concentration vs. time curve (AUC) for 3 hours after ingestion of the mixed meal.   
 
Statistical Analysis 
General principles.  Prior to all statistical analyses, the distributional properties of the outcome measures 
will be evaluated for normality (Shapiro-Wilks test) and equal variance (Levene’s test).  If the variables are 
not appropriately distributed, data transformations will be utilized to produce distributions that satisfy 
normality and equal variance assumptions.  If an appropriate transformation cannot be found, semi-
parametric methods based on the ranks of the data will be used; otherwise, parametric tests will be used.  
In the cross-sectional arm of the study, the primary goal will be to determine whether results obtained in 
MONW subjects differ from those obtained in metabolically-healthy subjects, and whether the differences 
between metabolic groups are modulated by ethnic background (Indian or Chinese) (Hypothesis 1).  
These goals will be accomplished by using analysis of variance with two between-subject factors.  The 
main effects of metabolic group and ethnicity, as well as the interaction between these two factors will be 
determined.  Statistically significant interactions, indicating that differences between metabolically healthy 
and unhealthy subjects vary by ethnicity, will be followed by Tukey’s post-hoc tests within each ethnic 
group.  In the interventional arm of the study, the primary goal will be to determine whether the changes 
induced by calorie restriction and exercise in the three intervention groups differ from the corresponding 
changes in the control group, as well as from each other (Hypothesis 2).  This will be accomplished by 
using repeated measures analysis of covariance, with the post-intervention value as the dependant 
variable, the intervention group as the independent variable, and the baseline value as a covariate.  In a 
secondary analysis, we will examine whether ethnicity modulates the response to diet or exercise, by 
using ethnic background (Indian or Chinese) as an additional between-subject factor.  This will be an 
exploratory aim, given our study is not adequately powered to address this question (we would need 
twice as many subjects in the intervention arm).  Nevertheless, out of the 20 subjects in each intervention 
group, we will recruit 10 from each ethnicity, and this will likely be enough to provide an indication of an 
ethnic-specific response to calorie restriction and exercise (if indeed any).  All these analyses will be 
preceded by preliminary between-group comparisons using Student’s unpaired t test (or the Mann-
Whitney U test), and χ2 test, to confirm there are no differences between groups with respect to key 
prognostic and demographic variables at baseline.  Although unlikely, given the planned balancing of the 
groups, any such variables that differ between groups at baseline will be adjusted for in subsequent 
analyses of covariance.  The fit of all models will be evaluated by assessing regression residuals.  We will 
accept statistical significance (type I error) at P ≤ 0.05.  Statistical analysis will be performed by using 
SPSS version 23 (IBM SPSS, Chicago, IL). 
 
Sample size considerations.  Our primary endpoint is whole-body insulin sensitivity (i.e. the major 
metabolic correlate of the MONW phenotype), determined by using the hyperinsulinemic-euglycemic 
clamp.  Previous studies that evaluated insulin-mediated glucose disposal in groups of healthy lean 
subjects on multiple occasions reported intra-individual coefficients of variation (CV) of 3–22 % [70-72].  
Under a conservative scenario, i.e. intra-individual CV of 21 % for insulin sensitivity, and an expected 
difference between metabolically healthy and unhealthy subjects in the order of ~25 % [22, 73], and an 
expected effect of mild weight loss in the order of ~20 % [51], we estimated we would need 14 subjects 
per group in the cross-sectional arm, and 9 subjects per group in the interventional arm, to detect 
statistically significant differences of this magnitude between metabolically healthy and unhealthy subjects 

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and before and after the interventions, respectively, using two-sided tests at the 5 % level of significance 
(i.e. type I error, α = 0.05) and with 80 % power (i.e. type II error, β = 0.20) [74].  Given that both of our 
hypotheses ultimately involve comparing different groups of people (whether comparing data at one point 
in time between two groups, as in Hypothesis 1; or comparing the change occurring between two points 
in time among four groups, as in Hypothesis 2), we decided to recruit 20 subjects per group in both the 
cross-sectional and the interventional arms of the study based on another conservative assumption, i.e. 
that only 70 % of all subjects will complete the study (n = 14 per group; dropout rate of 30%).  Based on 
our experience with similar lifestyle interventions leading to small amounts of weight loss [51], we expect 
much lower dropout rates (<25 %), so we are confident our study has more than adequate statistical 
power to meet its primary goals. 
 
Special considerations 
Definition of metabolic dysfunction.  Insulin resistance is the key metabolic abnormality of the MONW 
phenotype [7, 10, 24, 27], so the HOMA-IR score will be utilized to classify subjects as metabolically 
healthy or unhealthy at screening.  One might therefore expect that the hypothesized differences between 
groups in the M value and M/I ratio (Hypothesis 1) will be significant by definition.  However, the HOMA-
IR score is calculated from fasting plasma glucose and insulin concentrations [59] and is only a crude 
index that is not synonymous to (although it does correlate with) insulin sensitivity determined by the 
hyperinsulinemic-euglycemic clamp.  It may actually be more reflective of hepatic insulin sensitivity [75], 
whereas insulin-mediated glucose disposal determined during the clamp procedure is more reflective of 
muscle insulin sensitivity.  In addition, the cutoff values that will be used are based on the relationship 
between HOMA-IR and glucose tolerance [33], which is a function of both insulin sensitivity and insulin 
secretion.  These lines of reasoning support the argument that differences in HOMA-IR between groups 
(by design) cannot be considered equivalent to differences in insulin sensitivity, insulin secretion, or both.   
Definition of normal-weight.  The choice of a BMI <25 kg/m2 to indicate “leanness” may not be optimal 
for the Singaporean population, because of a varying relationship between BMI and percent body fat in 
Asian and Caucasian populations, but also among Asian populations [76, 77].  This implies that some 
subjects in the high-normal BMI range may actually be “overweight” by Asian standards.  Nevertheless, 
this will not affect interpretation of our results because metabolically healthy and unhealthy subjects will 
be matched for both BMI and percent body fat in this study.   
Subject retention.  No major problems with subject retention are expected, given the relatively modest 
intensity and short duration of the interventions.  Based on our experience [51], we anticipate 20-25 % of 
subjects will drop out or fail to comply with prescribed diet and exercise regimens.  However, the sample 
size was calculated based on a 30 % dropout rate to ensure an adequate number of subjects complete 
the study, even in the event of unanticipated greater dropout rates. 
Safety of study subjects.  This study involves consuming a low calorie diet or performing endurance 
exercise for 13-15 weeks.  In addition, subjects will undergo complex metabolic studies before and after 
the interventions.  Our research team has considerable experience in conducting exactly these types of 
studies, and has the clinical expertise to provide careful medical monitoring throughout the study to 
reduce the risk of any medical problems.  The research team will meet every week to review progress, 
problems and any clinical issues that arise during the study and either Dr. Leow or Dr. Toh will be 
available 24/7 on an on-call basis to address any medical problems that require urgent attention.  Also, 
we carefully designed the metabolic studies to ensure that the total amount of blood drawn from all 
research procedures is ~250 mL before and ~250 mL after the interventions, over a 4-month period. 
 

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