Original Article Obesity

CLINICAL TRIALS AND INVESTIGATIONS

Levels of Circulating miR-122 are Associated

with Weight Loss and Metabolic Syndrome
1, Lesli Hingstrup Larsen1, Pernille Bækgaard Udesen2,3, Yolanda Sanz4,
Anne Lundby Hess
Thomas Meinert Larsen1, and Louise Torp Dalgaard2

Objective: This study investigated whether the levels of specific serum

microRNAs (miRNAs) were altered following diet-induced weight loss Study Importance
and whether the serum miRNAs differed in the presence of the metabolic What is already known?
syndrome.
► Circulating miRNAs have been associ-
Methods: The study was a weight loss intervention trial with a prescribed ated with obesity and related metabolic
energy deficit of approximately 500 kcal/d. Levels of 22 miRNAs were abnormalities.
determined in serum samples from 85 participants with overweight or
obesity. miRNAs were analyzed using TaqMan Array miRNA Cards and What does this study add?
normalized to the geometric mean of spiked-in ath-miR-159a and U6 ► This study demonstrates that serum
small nuclear RNA using the ΔCT method. miR-122-5p, miR-126a-3p, miR-193a-
5p, and miR-222-3p change in response
Results: The average weight loss was 5.7 kg (P < 0.001). miR-122-5p to diet-induced weight loss.
(−0.18 ± 0.06 log fold relative to initial, P < 0.01) and miR-193a-5p ► Results suggest that circulating miR-
(−0.12 ± 0.04, P < 0.01) levels decreased in response to weight loss. 122-5p is an indicator of an adverse
miR-126a-3p (0.11 ± 0.04, P = 0.01) and miR-222-3p (1.51 ± 0.12, metabolic health status.
P < 0.001) levels increased. Furthermore, a higher level of miR-122-5p
was observed at baseline in participants with the metabolic syndrome
compared with participants without (0.28 ± 0.08, P < 0.01).
Conclusions: Changes in circulating miR-122-5p, miR-126a-3p,
miR-193a-5p, and miR-222-3p in response to diet-induced weight loss
are demonstrated. Furthermore, assessment of miR-122-5p could be an
indicator of an adverse metabolic health status independent of obesity.
Obesity (2020) 28, 493-501.

Introduction degradation and show a high degree of reproducibility within individu-

MicroRNA (miRNA) is a group of highly conserved, small noncod-
ing RNAs of approximately 22 nucleotides in size. miRNAs are able
to modulate gene expression through posttranscriptional regulation
by complementary binding to specific sites within target mRNAs (1).
Several studies have suggested that miRNAs are a significant contribu-
tor to the pathogenesis of complex diseases, including insulin resistance,
dyslipidemia, and cardiovascular diseases (2-4). From the cytoplasm,
mature miRNA can be released into circulation, yet exactly how circu-
lating miRNAs potentially influence cellular processes remains unclear.
However, it has been observed that alterations in circulating miRNA
profiles reflect the underlying physiological and pathological condi-
tions of the tissue (5). Circulating miRNAs are resistant to nuclease
als. This supports their potential as a tool to increase the understanding
of pathogenesis, as a target for clinical therapy, and as a noninvasive
diagnostic or progression biomarker in various diseases (5).
Several circulating miRNAs have been associated with obesity and met-
abolic disorders in humans. The first miRNA associated with a metabolic
phenotype was miR-122. This is a liver-enriched miRNA involved in the
regulation of cholesterol and lipid metabolism (6,7). Several studies have
reported that the expression level of circulating miR-122 is correlated
positively with BMI (8-10), and in a prospective, population-based study,
Willeit and colleagues reported that circulating miR-122 was associated
with future development of the metabolic syndrome (11).

1
Department of Nutrition, Exercise and Sports,  Faculty of Science,  University of Copenhagen, Frederiksberg, Denmark. Correspondence: Anne Lundby
Hess (lundbyhess@gmail.com) 2 Department of Science and Environment,  Roskilde University, Roskilde, Denmark 3 Department of Gynaecology and
Obstetrics, Fertility Clinic, Zealand University Hospital, Køge, Denmark 4 Microbial Ecology, Nutrition and Health Research Unit, Institute of Agrochemistry and
Food Technology, National Research Council (IATA-CSIC), Valencia, Spain.

© 2020 The Obesity Society. Received: 9 April 2019; Accepted: 14 October 2019; Published online 24 February 2020. doi:10.1002/oby.22704

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493
Obesity
Another circulating miRNA associated with obesity and the meta-
bolic syndrome is miR-221, which has been suggested to regulate
insulin resistance through its effect on adiponectin signaling in mice
(12). One study reported reduced levels of circulating miR-221 after
surgery-induced weight loss but not dietary-induced weight loss
(10). Lopez and colleagues demonstrated similar results; however,
the decrease in circulating miR-221 levels was present only in partic-
ipants after surgery-induced weight loss and not in participants after
surgery-induced weight loss following 6 months of exercise (13).
This indicates that circulating miR-221 levels, besides being affected
by extensive weight loss, may also be influenced by exercise. Similar
findings were demonstrated by Sawada and colleagues, who found a
decrease in circulating miR-221 levels as a response to acute resis-
tance exercise (14).
Randomized controlled trials investigating the association between
circulating miRNAs and diet-induced weight loss have not yielded
conclusive results regarding specific miRNAs. To explore the role of
circulating miRNAs in overweight, obesity, and the metabolic syn-
drome, we investigated whether the expression levels of selected
individual serum miRNA species were altered following hypocaloric
diet–induced weight loss and whether these specific miRNAs carry
potential as biomarkers of the metabolic syndrome.
Methods
Study design
The study (registered at ClinicalTrials.gov, identifier NCT03135041)
was a randomized, double-blinded, two-armed parallel intervention
trial that was conducted at the Department of Nutrition, Exercise and
Sports, University of Copenhagen. The study was conducted according
to the guidelines of the Declaration of Helsinki and conducted in accor-
dance with the standards of the Ethical Committee of the Capital Region
of Denmark (H-16045613) and the Danish Data Protection Agency
(2015-57-0116).
The participants were randomized to one of two dietary supplements
(fiber-containing dietary supplements or matched placebo) before
initiation of a 12-week energy-restricted weight loss period. The par-
ticipants attended a screening visit before the intervention period,
two clinical investigation days (one at baseline [week 0] and one at
the end of the intervention [week 12]), and five consultations with a
dietitian during the intervention with evaluation of body weight and
dietary compliance. The primary objective of the intervention trial
was to evaluate the effects of the fiber supplement on body weight
during energy restriction compared with the placebo supplement
(15). Analyses of circulating miRNAs were included as an explor-
atory outcome.
Study participants
The study participants were recruited through Web pages, social media,
and newspapers. The inclusion criteria required the participants, both
men and women, to be healthy, be between the ages of 18 and 60 years,
have BMI from 28 to 45 kg/m2, and have hemoglobin levels above
7.0 mmol/L. Exclusion criteria, which are reported elsewhere (15),
consisted of the use of medication for dyslipidemia, type 2 diabetes, or
elevated blood pressure; more than 10 hours of strenuous physical activ-
ity per week; and weight change of more than 3 kg within 2 months prior
to the study.
494     Obesity | VOLUME 28 | NUMBER 3 | MARCH 2020
Serum miR-122 Decreases in Response to Weight Loss  Hess et al.
Intervention
The fiber supplement consisted of 400 mL of milk containing 20 g
of prebiotic fiber (10 g inulin, Fibruline Instant; Cosruca, Warcoing,
Belgium) and 10 g of resistant maltodextrin (Fibersol-2; Matsutani,
Itami, Japan), and the placebo supplement contained 20 g of malto-
dextrin but was otherwise similar in content, taste, and appearance.
The participants were instructed to reduce their energy intake by ap-
proximately 500 kcal/d based on their calculated energy needs using
the Mifflin St. Jeor equation (16), including the incorporation of the
intervention products in the diet (contributing with 212 kcal/d), and to
consume an approximate macronutrient composition of 47 to 50 E%
carbohydrate, 32 to 33 E% fat, and 18 to 20 E% protein. A dietitian sys-
tematically guided the participants at five individual meetings during
the study period.
Clinical measurements
In the study, height was measured at the initial screening visit. Body
weight, waist and hip circumference, and sagittal height were measured
at both of the clinical investigation days. BMI was calculated as body
weight in kilograms divided by height in meters squared. Fat mass, lean
body mass, visceral adipose tissue, and fat percentage were determined at
the clinical investigation days by dual-energy x-ray absorptiometry scans
(Lunar Radiation iDXA; GE Healthcare, Chicago, Illinois). Venous blood
samples were drawn at the clinical investigation days after an overnight
fast. Total cholesterol, high-density lipoprotein cholesterol, triglycerides,
glucose, insulin, high-sensitivity C-reactive protein, aspartate amino-
transferase (ASAT), and alanine aminotransferase (ALAT) were ana-
lyzed at the Department of Nutrition, Exercise and Sports, University of
Copenhagen. The low-density lipoprotein cholesterol concentration was
calculated with the use of Friedewald’s equation (17). The homeostatic
model assessment of insulin resistance (HOMA-IR) was used to estimate
insulin resistance from measurements of fasting insulin and glucose con-
centrations. HOMA-IR was calculated as follows: (insulin [microunits per
milliliter] × glucose [millimoles per liter])/22.5. Blood pressure was mea-
sured with an automatically inflated cuff (A&D Instruments, Abingdon,
UK). Presence of criteria for the clinical diagnosis of the metabolic syn-
drome was determined according to the joint interim statement published
by the International Diabetes Federation, the American Heart Association,
the National Heart, Lung, and Blood Institute, and several other health or-
ganizations (18). The metabolic syndrome criteria are abdominal obesity,
elevated levels of triglycerides, reduced levels of high-density lipoprotein
cholesterol, hypertension, and elevated levels of fasting plasma glucose.
The presence of any three of these five risk factors constitutes the diagno-
sis of the metabolic syndrome.
Physical activity
Participants were asked to maintain their habitual level of physical
activity throughout the study. Prior to the two clinical investigation
days, the physical activity levels of the participants were measured for
seven consecutive days using a waist-worn accelerometer (GT3X+;
ActiGraph, Pensacola, Florida).
miRNA measurement
RNA was extracted from serum using TRIzol reagent and Phasemaker
tubes (ThermoFisher Scientific, Waltham, Massachusetts) according to
the manufacturer’s protocol. Spike-in RNA, ath-miR-159a (15nM), was
added to each sample during RNA extraction. The RNA concentration and
purity were determined using a NanoDrop ND-1000 spectrophotometer
(ThermoFisher Scientific). Reverse transcription, preamplification, and
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Original Article
CLINICAL TRIALS AND INVESTIGATIONS
real-time polymerase chain reaction (PCR) of the plasma miRNA were
conducted using reverse transcription reaction mix, PreAmp reaction mix,
and TaqMan Universal PCR Master Mix (ThermoFisher Scientific) ac-
cording to the manufacturer’s protocol. miRNA expression was analyzed
using custom TaqMan Array miRNA Cards containing 22 miRNAs of in-
terest, selected from the literature, and 2 controls (Supporting Information
Tables S1-S2). Samples before and after the intervention period for
every participant were added to the same card to minimize plate biases.
Expression data were obtained using the ViiA 7 Real-Time PCR System
(ThermoFisher Scientific) according to the manufacturer’s protocol. Data
were normalized to the geometric mean of spike-in control ath-miR-159a
and the endogenous control U6 small nuclear RNA. This was determined
as a homogenous and stable control with the geometric mean cycle thresh-
old (CT) of 17.7 ± 2.7 at baseline and 17.4 ± 2.4 after weight loss. Fold
changes were expressed relative to either baseline, no presence of the met-
abolic syndrome, or presence of one criteria of the metabolic syndrome,
depending on the actual statistical analysis. This was done with the use
of the ΔΔCT method (19). miRNAs in samples with a CT level higher
than 32, determined by the technical characteristics of the platform (ViiA
7 Real-Time PCR System), were excluded before further analysis. miR-
208 was not amplified in any of the samples. miR-589 was amplified in a
few samples with a mean CT of 32.5 ± 0.8. Both miRNAs were therefore
excluded from further analyses.
Statistical analyses
No effect of the fiber supplement on body weight during energy re-
striction compared with the placebo supplement was observed (15).
Therefore, data from the two groups were pooled to achieve the ob-
jective of this article. Normality of distribution for quantitative data
was determined using Shapiro-Wilk tests. Expression of miRNAs
was logarithmically transformed before statistical analyses. Mean
fold changes with associated SEs were used for summarizing sig-
nificant main effects. Analyses of the levels of circulating miRNAs
and the relationship with diet-induced weight loss or the metabolic
syndrome used linear models with adjustment for sex, age, physical
activity, and type of dietary supplement. The Spearman correlation
coefficient was used to assess correlations between miRNAs and rel-
evant metabolic variables. A value of P ≤ 0.05 was considered statis-
tically significant. Each miRNA chosen for analysis was based on
results from prior research (Supporting Information Tables S1-S2),
and data are presented as nominal and Bonferroni-adjusted P val-
ues. All of the statistical analyses were performed using R version
3.5.1 (R Foundation for Statistical Computing, Vienna, Austria).
Results
Baseline characteristics and weight loss
We included 85 participants in the analysis. The participants had
a mean  BMI of 33.8 (SD  4.2) at baseline and 31.8 (SD  4.2) after the
weight loss intervention. All of the indices of the metabolic syndrome
changed significantly from baseline to the  end of the intervention
(Table 1).
Change in circulating miRNA levels after
diet-induced weight loss
Four of the twenty circulating miRNAs changed significantly in re-
sponse to weight loss (Figure 1). The levels of miR-122-5p and miR-
193a-5p were reduced with mean log-fold changes of −0.18 ± 0.06
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Obesity
(P = 0.003, adjusted P = 0.06) and −0.12 ± 0.04 (P = 0.004, adjusted
P = 0.08), respectively. miR-126a-3p and miR-222-3p levels were
increased from pre- to postintervention, with mean fold changes of
0.11 ± 0.04 (P = 0.014, adjusted P = 0.80) and 1.51 ± 0.12 (P < 0.001,
adjusted P = 0.02), respectively. The remaining 16 miRNAs did not
change in response to weight loss (Supporting Information Figure S1).
Circulating miRNA levels and metabolic
syndrome
At baseline, all of the participants had at least one criteria of the
metabolic syndrome because they all had abdominal obesity (waist
circumference ≥ 80 cm). The number and distribution of the different
criteria among the participants can be seen in Supporting Information
Figure S2. In total, 45 of the 85 participants (52.9%) had the meta-
bolic syndrome at baseline according to the joint interim statement
published by the International Diabetes Federation, the American
Heart Association, the National Heart, Lung, and Blood Institute, and
several other health organizations (18). The levels of miR-122-5p and
miR-193a-5p were higher in the presence of the metabolic syndrome,
with mean log-fold differences of 0.28 ± 0.08 (P = 0.001, adjusted
P = 0.02) and 0.13 ± 0.06 (P = 0.03, adjusted P = 0.61), respectively.
Additionally, miR-485-5p levels were lower, with a mean fold dif-
ference of −0.42 ± 0.15 (P = 0.010, adjusted P = 0.2) (Figure 2). There
were no significant differences in the remaining circulating miRNA
levels between participants with and without the metabolic syn-
drome (Supporting Information Figure S3). After the diet-induced
weight loss, participants with the metabolic syndrome were reduced
to 44.7%. None of the tested circulating miRNA levels was signifi-
cantly different between participants with and without the metabolic
syndrome after the weight loss intervention.
Further analyses of the distribution of the metabolic syndrome crite-
ria revealed an increasing trend in the level of circulating miR-122-5p
according to the number of metabolic syndrome–related criteria present
(Figure 3). Participants characterized with two criteria of the metabolic
syndrome had significantly lower levels of circulating miR-122-5p
compared with participants with three (P = 0.014), four (P = 0.024), or
five (P = 0.027) criteria present.
Correlations between miRNAs and clinical
variables
As the levels of circulating miR-122-5p, miR-126-3p, miR-193a-5p,
miR-222-3p, and miR-485-5p changed significantly during the weight
loss intervention and/or because of presence of the metabolic syn-
drome, correlation analyses between these miRNAs and specific meta-
bolic variables were conducted (Table 2).
miR-122-5p levels correlated positively with several markers of body
composition at baseline: body weight, waist circumference, sagittal
height, lean body mass, and, in particular, visceral adipose tissue (ρ = 0.47,
P < 0.001). The correlation with sagittal height (ρ = 0.27, P = 0.023), lean
body mass (ρ = 0.30, P = 0.012), and visceral adipose tissue (ρ = 0.32,
P = 0.006) was further confirmed because a decrease from pre- to postin-
tervention was correlated with a decrease in the expression levels of miR-
122-5p. At baseline, miR-122-5p also correlated positively with insulin
(ρ = 0.41, P < 0.001) and HOMA-IR (ρ = 0.41, P < 0.001). However, these
correlations were not observed when assessing correlations over time.
Furthermore, circulating miR-122-5p was positively correlated with
ASAT (ρ = 0.48, P < 0.001), ALAT (ρ = 0.58, P < 0.001), and hypertension
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Obesity Serum miR-122 Decreases in Response to Weight Loss  Hess et al.

TABLE 1 Characteristics of study participants

Baseline, After weight loss, Mean (95% CI) of

  mean ± SD mean ± SD difference P
N (women/men) 85 (55/30)      
Body composition        
Age (y) 48.5 ± 8.8      
Height (cm) 1.72 ± 0.10      
Body weight (kg) 99.5 ± 16. 8 93.8 ± 16.7 5.70 (4.94 to 6.45) < 0.001
BMI 33.8 ± 4.2 31.8 ± 4.2 1.95 (1.69 to 2.20) < 0.001
Waist circumference (cm) 106.6 ± 11.5 101.0 ± 12.0 5.52 (4.53 to 6.51) < 0.001
Hip circumference (cm) 117.7 ± 9.4 113.0 ± 9.45 4.67 (3.90 to 5.43) < 0.001
Sagittal height (cm) 25.4 ± 2.85 23.5 ± 3.0 1.97 (1.67 to 2.26) < 0.001
Fat mass (kg) 41.7 ± 9.5 37.1 ± 9.9 4.52 (3.86 to 5.18) < 0.001
Lean body mass (kg) 54.3 ± 11.9 53.4 ± 11.8 0.94 (0.67 to 1.22) < 0.001
Visceral adipose tissue (kg) 1.97 ± 1.09 1.61 ± 0.95 0.35 (0.28 to 0.43) < 0.001
Fat percentage (%) 42.3 ± 6.9 39.8 ± 7.6 2.49 (2.02 to 2.96) < 0.001
Lipid profile        
Total cholesterol (mmol/L) 5.25 ± 0.93 4.96 ± 0.90 0.30 (0.16 to 0.43) < 0.001
HDL cholesterol (mmol/L) 1.31 ± 0.30 1.26 ± 0.30 0.05 (0.01 to 0.10) 0.031
LDL cholesterol (mmol/L) 3.27 ± 0.87 3.09 ± 0.76 0.17 (0.05 to 0.30) 0.008
Triglycerides (mmol/L) 1.49 ± 0.94 1.34 ± 0.72 0.15 (0.03 to 0.28) 0.019
Glucose metabolism        
Glucose (mmol/L) 5.75 ± 0.95 5.54 ± 0.45 0.22 (0.05 to 0.38) 0.011
Insulin (pmol/L) 106.8 ± 73.2 88.1 ± 64.1 18.7 (7.3 to 30.1) 0.002
HOMA-IR 4.05 ± 3.05 3.18 ± 2.42 0.87 (0.37 to 1.37) < 0.001
Inflammation markers        
hsCRP (mg/L) 4.67 ± 7.34 4.11 ± 6.81 0.56 (−1.08 to 2.19) 0.502
ASAT (U/L) 28.0 ± 14.4 24.2 ± 10.7 3.79 (1.53 to 6.04) 0.001
ALAT (U/L) 34.1 ± 25.9 25.4 ± 12.9 8.77 (3.80 to 13.7) < 0.001
Blood pressure        
Systolic (mmHg) 125.3 ± 14.8 121.7 ± 14.9 3.63 (1.44 to 5.82) 0.001
Diastolic (mmHg) 83.5 ± 10.5 81.1 ± 9.8 2.43 (0.88 to 3.97) 0.002
Other        
Physical activity (CPM) 605.3 ± 158.8 630.7 ± 148.9 −15.2 (−43.7 to 13.3) 0.291

Mean ± SD for outcomes at baseline and after 12 weeks of energy restriction. Difference between outcomes at baseline and after 12 weeks were analyzed with Student paired
t test. Bold font indicates significant P values.
CPM, counts per minute; HDL, high-density lipoprotein; hsCRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein; HOMA-IR, homeostatic model assessment of
insulin resistance; ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase.

(systolic: ρ = 0.33, P = 0.005; diastolic: ρ = 0.36, P = 0.002) at baseline.
Likewise, the change in miR-122-5p levels was positively correlated with
weight loss–induced changes in ASAT (ρ = 0.49, P < 0.001) and ALAT
levels (ρ = 0.51, P < 0.001) but not with hypertension (systolic: ρ = 0.23,
P = 0.057; diastolic: ρ = 0.22, P = 0.065).
miR-126-3p correlated positively with body weight, waist circumference,
lean body mass, and visceral adipose tissue and correlated negatively with
body fat percentage at baseline. An increase in circulating miR-126-3p
levels correlated with a decrease in insulin levels (ρ = −0.23, P = 0.032)
and HOMA-IR (ρ = −0.23, P = 0.033) from pre- to postintervention.
miR-193a-5p correlated positively with several markers of body com-
position at baseline: body weight, BMI, waist circumference, sagittal
height, fat mass, lean body mass, and visceral body mass. Only the
correlation with lean body mass was present during the intervention
because a positive change in miR-193a-5p correlated with an increase
in lean body mass (ρ = 0.34, P = 0.009). miR-193a-5p further correlated
positively with insulin (ρ = 0.38, P = 0.002), HOMA-IR (ρ = 0.33,
P = 0.002), ASAT (ρ = 0.31, P = 0.011), and ALAT (ρ = 0.29, P = 0.020)
at baseline. The correlation with ASAT was further present from pre- to
postintervention (ρ = 0.28, P = 0.028). Finally, an increase in circulat-
ing miR-193a-5p levels was correlated with diastolic blood pressure
(ρ = 0.29, P = 0.025) from pre- to postintervention.
No significant correlations, neither at baseline nor during the weight
loss intervention, were observed for circulating miR-222-3p and the
included metabolic variables.

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Original Article Obesity
CLINICAL TRIALS AND INVESTIGATIONS

Figure 1  Box  plot illustrating the miRNA levels at baseline and after 12 weeks of energy restriction for all participants. Data were normalized to the
geometric mean of ath-miR-159 and U6 small nuclear RNA (y-axis) and presented as mean log-fold changes relative to baseline (y-axis). miR-122-
5p (n = 74), −0.18 ± 0.06, P = 0.003. miR-126a-3p (n = 85), 0.11 ± 0.04, P = 0.014. miR-193a-5p (n = 66), −0.12 ± 0.04, P = 0.004. miR-222-3p (n = 80),
1.51 ± 0.12, P < 0.001. miRNA data were log transformed before analyses. Difference was analyzed with linear models adjusted for age, sex, physical
activity, and dietary supplement.

miR-485-5p correlated positively with fat mass (ρ = 0.42, P = 0.028)
and visceral adipose tissue (ρ = 0.43, P = 0.025) at baseline. The cor-
relations were also present during the intervention; however, in this
case, positive changes in miR-485-5p correlated with a decrease in
fat mass (ρ = −0.64, P = 0.010) and visceral adipose tissue (ρ = −0.59,
P = 0.019). Furthermore, a positive change in miR-485-5p correlated
with a decrease in BMI, sagittal height, fat percentage, and triglyceride
levels from pre- to postintervention.
S1-S2). These miRNAs were investigated in a large randomized controlled
diet-induced weight loss study to explore their role in weight loss and their
potential as biomarkers of the presence of the metabolic syndrome.
We identified a significant change in levels of four of the included miR-
NAs after the weight loss intervention. The levels of both miR-122-5p
and miR-193a-5p were upregulated, whereas miR-126a-3p and miR-
222-3p levels were downregulated after the diet-induced weight loss
compared with baseline.

Effect of fiber supplementation on circulating
miRNAs
We observed a difference from pre- to postintervention between the
groups receiving a placebo or fiber supplementation for miR-16-5p
(Supporting Information Figure S4). The level of circulating miR-16-5p
increased with a mean log-fold change of 0.16 ± 0.07 in response to fiber
supplementation compared with a placebo (P = 0.021, adjusted P = 0.42).
Discussion
Although the exact mechanism of the release of circulating miRNAs
is largely unknown, several studies have highlighted their potential as
predictive and reflective markers of disease development.
Through a literature review, we selected 22 specific circulating miRNAs
reported to relate to overweight and obesity (Supporting Information Tables
The reduced level of circulating miR-122-5p in the participants after the
intervention could indicate improved liver health after weight loss. The
reasoning is supported by the strong positive correlation between circu-
lating miR-122-5p and the liver enzymes ASAT and ALAT observed at
baseline and during the intervention. In 2002, miR-122 was identified
as an abundant miRNA in the liver through cloning and sequencing
of small RNA prepared from different mouse tissues (20). Later, miR-
122 was found highly expressed in human primary hepatocytes (21).
Silencing of miR-122 in high-fat diet–fed mice resulted in significant
reduction of hepatic steatosis, which was associated with reduced cho-
lesterol synthesis rates and stimulation of hepatic fatty-acid oxidation
(6,22). In contradiction to this, and to other studies (9,11), however, we
did not observe significant correlations between miR-122 and blood
cholesterol levels. However, circulating levels of miR-122 were posi-
tively correlated with triglycerides at baseline. The significant positive
correlation between the change in miR-122-5p and the change in vis-
ceral adipose tissue and blood pressure, along with the results regarding

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Obesity Serum miR-122 Decreases in Response to Weight Loss  Hess et al.

Figure 2 Box plot of baseline miRNAs for participants with and without metabolic syndrome (characterized by the joint interim statement). Data were
normalized to the geometric mean of ath-miR-159 and U6 small nuclear RNA (y-axis) and presented as mean log-fold changes relative to no presence
of the metabolic syndrome (y-axis). miR-122-5p (n = 36 without metabolic syndrome/38 with metabolic syndrome): 0.28 ± 0.08, P = 0.001; miR-193a-5p
(n = 30/36): 0.13 ± 0.06, P = 0.030; miR-485-5p (n = 13/15): −0.42 ± 0.15, P = 0.010. miRNA data were log transformed before analyses. Difference was
analyzed with linear models adjusted for age, sex, and physical activity.

ASAT and ALAT, could suggest a negative correlation between miR-
122-5p and improved metabolic health. We demonstrated a potential
of miR-122-5p as constituting a biomarker of the presence of the met-
abolic syndrome as the level of circulating miR-122-5p increased with
the number of metabolic syndrome criteria present, and the level was
significantly higher in participants classified with the metabolic syn-
drome compared with participants without the metabolic syndrome.
These results are in line with the results reported by Willeit and col-
leagues (11). The results suggest a possible diagnostic use of circulating
miR-122-5p in the characterization of the progression of the metabolic
syndrome, thereby determining whether individuals are metabolically
unhealthy independent of the degree of obesity. Our study findings sup-
port a recent systematic review and meta-analysis on the circulatory
levels of miR-122 in relation to nonalcoholic fatty liver disease showing
a general upregulation of circulating miR-122 levels in nonalcoholic
fatty liver disease (23), which supports our notion that decreased miR-
122 levels following weight loss could reflect improved liver steatosis.
However, further studies are necessary to further evaluate whether mea-
surement of circulating miR-122 levels could benefit current methods
of establishing metabolically unhealthy overweight or obesity.
Only a few studies have reported significant findings on circulating miR-
193a-5p in relation to obesity. One study observed that surgery-induced
weight loss led to a marked decrease of circulating miR-193a-5p levels
but was not able to demonstrate the same tendency with diet-induced
weight loss (10). In the present study, we observed a significant decrease
in the levels of circulating miR-193a-5p after the weight loss interven-
tion. Furthermore, the circulating miR-193a-5p levels were higher in
participants with the metabolic syndrome than in those without the meta-
bolic syndrome. An in vivo study observed an increase in the expression
of miR-193a-5p in skeletal muscle in participants with type 2 diabetes
compared with participants with normal glucose tolerance (24). Similarly
to miR-122, miR-193a-5p is liver enriched, which is in accordance with
the correlations in circulating levels of miR-122 and miR-193a-5p
(Supporting Information Table S3). Because the change in miR-193a-5p
levels was shown to correlate positively with the change in glucose con-
centration in our study, the decrease of the miR-193a-5p ­levels after
weight loss might be related to an improved glucose metabolism.
In the present study, we observed an increase in circulating levels of
miR-126a-3p and miR-222-3p after the weight loss intervention. Both
miR-126a-3p and miR-222-3p have been linked to the regulation of glu-
cose metabolism. An in vitro study in human hepatocytes demonstrated
that miR-126 is a crucial inhibitory factor in insulin signal transduc-
tion because it represses translation of insulin receptor substrate-1 (25).
Furthermore, a cross-sectional study reported significant differences in
the two miRNAs between participants with and without type 2 diabetes.
Circulating levels of miR-126a-3p were lower and those of miR-222-3p
were higher in participants with type 2 diabetes compared with partici-
pants with normal glucose tolerance (26). However, we did not observe
correlations between the circulating miRNAs and markers of glucose
metabolism in the present study. Moreover, miR-126-3p is highly

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Original Article Obesity
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Figure 3 Box plot of miR-122-5p at baseline for participants with one to five criteria used to define metabolic syndrome according to the joint interim
statement. Data were normalized to the geometric mean of ath-miR-159 and U6 small nuclear RNA and presented as log-fold change relative to
the presence of one criteria of metabolic syndrome. Significant differences are found between participants with one and five criteria of the metabolic
syndrome (0.40 ± 0.19, P = 0.043), two and three criteria (0.25 ± 0.10, P = 0.014), two and four criteria (0.29 ± 0.13, P = 0.024), and two and five criteria
(0.41 ± 0.18, P = 0.027). Differences are determined through linear regression analysis with adjustment of age, sex, and physical activity. Data on miRNAs
were log transformed before statistical analyses.

enriched in endothelial cells from lymphoid vessels, blood vessels,
and liver sinusoids. miR-222-3p is more widely expressed but is also
expressed at high levels in endothelial cell types (27), and circulating
levels of miR-126a-3p and miR-222-3p are also significantly correlated
(Supporting Information Table S3). It is possible that the increase in
miR-126-3p and miR-222-3p levels following weight loss could reflect
improved endothelial function following reduction of low-grade inflam-
mation, but to address this question further, studies are necessary.
miR-485-5p is expressed in the human liver and has been reported to
target genes involved in lipid metabolism (28). Our results on circulat-
ing miR-485-5p in relation to weight loss and the metabolic syndrome
are not clear, and to specify the function of this miRNA in circulation,
more research has to be conducted. We also identified the fact that
miR-16-5p levels are modulated by fiber supplementation. This has,
to our knowledge, not been reported before. The mechanism for this
regulation is unknown, but because miR-16-5p is enriched in cells of
the immune system (B cells, monocytes) (27), the mechanism could
be related to the circulating or intestinal levels of immune-regulatory
short-chain fatty acids because these have previously been shown to
be increased following the intake of prebiotic dietary fiber (29).
Even though recent studies have suggested the potential of circulating
miRNAs as tools to increase the understanding of obesity-associated
metabolic complications, we were only able to relate 5 of 20 tested
miRNAs to weight loss or other variables related to intermedi-
ary metabolism, although the tested miRNAs had previously been
reported to be associated with these phenotypes. The reason for this
discrepancy is not known but could be due to previous reports being
based on cohorts with very low numbers (less than 20 participants
per group), resulting in increased risk of false-positive findings.
Another source of variability might be the diversity in the designs
of the current studies reporting on circulating miRNAs in relation to
overweight and obesity (Supporting Information Table S2). The lon-
gitudinal and randomized design of our study gives it stronger power
to detect changes in circulating miRNA levels in relation to weight
loss. However, the moderate weight loss and period of follow-up in
our study was perhaps not sufficient to alter these miRNAs to a degree
necessary to elicit changes in circulating levels, despite our observa-
tions of an improved metabolic health (Table 1). Thus, further vali-
dation studies are warranted. Furthermore, the discrepancy could be
due to different measurement methods for different reported studies,
for example, type of biofluids, as reported in Supporting Information
Table S2. For the research field of miRNAs in circulation, standard-
ized strategies of reporting are yet to be formulated and agreed on. It is
important to address the strategy regarding analytical factors affecting
measurement, including standardization of sample type, measurement
platform, and normalization strategy, before it is possible to directly

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TABLE 2 Correlations between selected miRNAs and specific metabolic variables

miR-122-5p miR-126a-3p miR-193a-5p miR-222-3p miR-485-5p

Baseline Change Baseline Change Baseline Change Baseline Change Baseline Change

  ρ P ρ P ρ P ρ P ρ P ρ P ρ P ρ P ρ P ρ P
Body composition                                        
Body weight (kg) 0.28 0.014 0.20 0.095 0.22 0.046 −0.05 0.679 0.32 0.008 0.11 0.397 −0.05 0.632 0.04 0.721 0.28 0.153 −0.58 0.018
BMI 0.08 0.523 0.15 0.218 0.07 0.551 −0.04 0.705 0.28 0.024 0.10 0.465 −0.10 0.394 0.05 0.641 0.36 0.064 −0.48 0.062
Waist circumference (cm) 0.28 0.014 0.07 0.553 0.25 0.022 −0.03 0.782 0.30 0.016 0.01 0.935 −0.03 0.819 0.07 0.515 0.30 0.120 −0.32 0.232

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Hip circumference (cm) −0.11 0.360 0.04 0.763 −0.10 0.357 −0.21 0.059 0.12 0.323 0.09 0.490 −0.15 0.198 −0.08 0.475 0.28 0.156 −0.26 0.334
Sagittal height (cm) 0.34 0.004 0.27 0.023 0.15 0.180 −0.06 0.584 0.38 0.002a  0.15 0.251 −0.09 0.449 0.05 0.674 0.35 0.071 −0.55 0.027
Fat mass (kg) 0.14 0.226 0.18 0.121 −0.01 0.956 −0.04 0.709 0.30 0.015 0.13 0.305 −0.12 0.278 0.04 0.746 0.42 0.028 −0.64 0.010
Lean body mass (kg) 0.27 0.023 0.30 0.012 0.29 0.008 −0.07 0.518 0.24 0.049 0.34 0.009 −0.01 0.922 0.07 0.567 0.05 0.816 0.15 0.586
Visceral adipose tissue (kg) 0.47 < 0.001a  0.32 0.006 0.29 0.008 0.06 0.575 0.36 0.003 0.20 0.115 −0.04 0.696 0.05 0.670 0.43 0.025 −0.59 0.019
Fat percent (%) −0.12 0.328 0.12 0.328 −0.27 0.012 −0.04 0.750 0.03 0.843 0.07 0.571 −0.10 0.400 0.03 0.814 0.25 0.195 −0.64 0.010
Lipid profile                                        
Total cholesterol (mmol/L) 0.13 0.261 0.02 0.887 0.01 0.956 −0.12 0.273 −0.05 0.670 −0.03 0.829 −0.06 0.572 −0.18 0.123 0.14 0.486 −0.36 0.176
HDL cholesterol (mmol/L) −0.15 0.207 0.02 0.899 −0.16 0.153 0.08 0.478 −0.11 0.391 0.06 0.641 0.05 0.673 0.07 0.526 0.16 0.423 −0.30 0.261
LDL cholesterol (mmol/L) 0.01 0.966 −0.07 0.569 0.03 0.802 −0.09 0.397 −0.10 0.430 −0.13 0.315 −0.11 0.325 −0.17 0.147 0.18 0.358 −0.04 0.900
Triglycerides (mmol/L) 0.47 < 0.001a  0.08 0.495 0.13 0.237 −0.14 0.189 0.20 0.118 0.19 0.139 0.05 0.687 −0.09 0.413 −0.06 0.769 −0.53 0.037
Glucose metabolism                                        
Glucose (mmol/L) 0.14 0.220 −0.14 0.251 −0.15 0.181 −0.10 0.387 0.16 0.188 0.04 0.756 −0.20 0.076 −0.10 0.378 0.08 0.680 −0.24 0.377
Insulin (pmol/L) 0.41 < 0.001a  0.01 0.916 0.04 0.717 −0.23 0.032 0.38 0.002a  0.02 0.878 −0.09 0.445 −0.17 0.135 0.04 0.836 −0.19 0.492
HOMA-IR 0.41 < 0.001a  −0.03 0.803 0.02 0.839 −0.23 0.033 0.37 0.002a  0.04 0.778 −0.10 0.395 −0.19 0.098 0.04 0.860 −0.24 0.379
Inflammation markers                                        
hsCRP (mg/L) 0.05 0.653 0.02 0.848 0.16 0.151 −0.13 0.228 0.19 0.120 0.00 0.976 0.08 0.490 −0.11 0.340 0.37 0.052 −0.12 0.656
ASAT (U/L) 0.48 < 0.001a  0.49 < 0.001a  0.13 0.229 0.08 0.456 0.31 0.011 0.28 0.028 −0.08 0.495 0.25 0.027 0.14 0.465 −0.33 0.218
ALAT (U/L) 0.58 < 0.001a  0.51 < 0.001a  0.11 0.334 0.02 0.876 0.29 0.020 0.17 0.197 −0.08 0.536 0.13 0.268 −0.15 0.459 −0.32 0.224
Blood pressure                                        
Systolic (mmHg) 0.33 0.005 0.23 0.057 0.06 0.602 0.07 0.516 0.08 0.514 0.20 0.130 0.08 0.497 0.20 0.071 −0.15 0.459 −0.06 0.837
Diastolic (mmHg) 0.36 0.002a  0.22 0.065 0.00 0.974 0.06 0.608 0.09 0.464 0.29 0.025 0.09 0.423 0.24 0.035 −0.15 0.449 −0.45 0.080

aSignificantafter adjustment for multiple comparisons using Bonferroni.

Spearman correlations (ρ) between miRNA levels and variables at baseline and correlations between change in miRNA levels and variables from pre- to postintervention. Data for miRNAs were normalized to geometric mean
of ath-miR-159 and U6 small nuclear RNA and relative to baseline. Bold font indicates significant P values.
ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; HDL, high-density lipoprotein; hsCRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein; HOMA-IR, homeostatic model assessment of insulin
resistance.
Serum miR-122 Decreases in Response to Weight Loss  Hess et al.

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Original Article
CLINICAL TRIALS AND INVESTIGATIONS
compare studies and to conclude that circulating miRNAs can be used
as reliable biomarkers.
In conclusion, this study demonstrated a change in circulating
miR-122-5p, miR-126a-3p, miR-193a-5p, and miR-222-3p levels in
response to a diet-induced weight loss intervention. Furthermore, our
results suggest that an assessment of miR-122 could indicate metabolic
health status independently of obesity. O
12. Lustig Y, Barhod E, Ashwal-Fluss R, et al. RNA-binding protein PTB and mi-
Acknowledgments
The authors wish to thank the participants and the study staff (scien-
tific employees, dietitians, kitchen staff, laboratory technicians, and
bachelor’s and master’s students) involved in the intervention study
at the Department of Nutrition, Exercise and Sports, University of
Copenhagen. Additionally, thanks to CAPSA FOOD (partner in the
European Union’s Seventh Framework Program) for providing the di-
etary supplements.
We commit to responsible sharing of data from this clinical trial.
This includes summary data and anonymized individual participant
data as well as other information, such as study protocol. Requests
from any qualified researchers who engage in rigorous, independent
scientific research will be considered. Data will be provided follow-
ing review and approval of a research proposal and statistical analysis
plan and execution of a data sharing agreement. For more information,
please email Thomas Meinert Larsen, tml@nexs.ku.dk.
Funding agencies: This work was supported by the European Union’s Seventh
Framework Program, grant 613979 (MyNewGut), and by the European Union/
European Federation of Pharmacutical Industries and Associations (EU/EFPIA)
Innovative Medicines Initiative Joint Undertaking European Medical Information
Framework (EMIF) grant 115372. PBU is funded by ReproUnion, cofinanced by the
European Union and Interreg V ÔKS.
Disclosure: The authors declared no conflict of interest.
Clinical trial registration: ClinicalTrials.gov identifier NCT03135041.
Supporting Information: Additional Supporting Information may be found in the
online version of this article.
References
1. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved
targets of microRNAs. Genome Res 2009;19:92-105.
2. Hashimoto N, Tanaka T. Role of miRNAs in the pathogenesis and susceptibility of dia-
betes mellitus. J Hum Genet 2017;62:141-150.
3. Flowers E, Aouizerat BE. MicroRNA associated with dyslipidemia and coronary dis-
ease in humans. Physiol Genomics 2013;45:1199-1205.
4. Romaine SP, Tomaszewski M, Condorelli G, Samani NJ. MicroRNAs in cardiovascular
disease: an introduction for clinicians. Heart 2015;101:921-928.
5. Velu VK, Ramesh R, Srinivasan AR. Circulating MicroRNAs as biomarkers in health
and disease. J Clin Diagn Res 2012;6:1791-1795.
www.obesityjournal.org 
Obesity
6. Krutzfeldt J, Rajewsky N, Braich R, et al. Silencing of microRNAs in vivo with ‘antag-
omirs’. Nature 2005;438:685-689.
7. Elmén J, Lindow M, Schütz S, et al. LNA-mediated microRNA silencing in non-human
primates. Nature 2008;452:896-899.
8. Ameling S, Kacprowski T, Chilukoti RK, et al. Associations of circulating plasma
microRNAs with age, body mass index and sex in a population-based study. BMC Med
Genomics 2015;8:61. doi:10.1186/s12920-015-0136-7
9. Wang R, Hong J, Cao Y, et al. Elevated circulating microRNA-122 is associated with
obesity and insulin resistance in young adults. Eur J Endocrinol 2015;172:291-300.
10. Ortega FJ, Mercader JM, Catalán V, et al. Targeting the circulating microRNA signature
of obesity. Clin Chem 2013;59:781-792.
11. Willeit P, Skroblin P, Moschen AR, et al. Circulating microRNA-122 is associ-
ated with the risk of new-onset metabolic syndrome and type 2 diabetes. Diabetes
2017;66:347-357.
croRNA-221 coregulate AdipoR1 translation and adiponectin signaling. Diabetes
2014;63:433-445.
13. Nunez Lopez YO, Coen PM, Goodpaster BH, Seyhan AA. Gastric bypass surgery with
exercise alters plasma microRNAs that predict improvements in cardiometabolic risk.
Int J Obes (Lond) 2017;41:1121-1130.
14. Sawada S, Kon M, Wada S, Ushida T, Suzuki K, Akimoto T. Profiling of circulating mi-
croRNAs after a bout of acute resistance exercise in humans. PLoS One 2013;8:e70823.
doi:10.1371/journ​al.pone.0070823
15. Hess AL, Benítez-Páez A, Blædel T, et al. The effect of inulin and resistant malto-
dextrin on weight loss during energy restriction: a randomised, placebo-controlled,
double-blinded intervention [published online October 11, 2019]. Eur J Nutr 2019.
doi:10.1007/s00394-019-02099-x
16. Mifflin MD, St Jeor ST, Hill LA, Scott BJ, Daugherty SA, Koh YO. A new predic-
tive equation for resting energy expenditure in healthy individuals. Am J Clin Nutr
1990;51:241-247.
17. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-den-
sity lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.
Clin Chem 1972;18:499-502.
18. Alberti KG, Eckel RH, Grundy SM, et al. Harmonizing the metabolic syndrome: a joint
interim statement of the International Diabetes Federation Task Force on Epidemiology
and Prevention; National Heart, Lung, and Blood Institute; American Heart Association;
World Heart Federation; International Atherosclerosis Society; and International
Association for the Study of Obesity. Circulation 2009;120:1640-1645.
19. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR.
Nucleic Acids Res 2001;29:e45. doi:10.1093/nar/29.9.e45
20. Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T. Identification
of tissue-specific microRNAs from mouse. Curr Biol 2002;12:735-739.
21. Chang J, Nicolas E, Marks D, et al. miR-122, a mammalian liver-specific microRNA, is
processed from hcr mRNA and may downregulate the high affinity cationic amino acid
transporter CAT-1. RNA Biol 2004;1:106-113.
22. Esau C, Davis S, Murray SF, et al. miR-122 regulation of lipid metabolism revealed by
in vivo antisense targeting. Cell Metab 2006;3:87-98.
23. Cai C, Lin Y, Yu C. Circulating miRNAs as novel diagnostic biomarkers in nonalcoholic
fatty liver disease: a systematic review and meta-analysis. Can J Gastroenterol Hepatol
2019;2019:2096161. doi:10.1155/2019/2096161
24. Gallagher IJ, Scheele C, Keller P, et al. Integration of microRNA changes in vivo iden-
tifies novel molecular features of muscle insulin resistance in type 2 diabetes. Genome
Med 2010;2:9. doi:10.1186/gm130​
25. Ryu HS, Park SY, Ma D, Zhang J, Lee W. The induction of microRNA targeting IRS-1
is involved in the development of insulin resistance under conditions of mitochondrial
dysfunction in hepatocytes. PLoS One 2011;6:e17343. doi:10.1371/journ​ al.pone.
0017343
26. Ortega FJ, Mercader JM, Moreno-Navarrete JM, et al. Profiling of circulating microR-
NAs reveals common microRNAs linked to type 2 diabetes that change with insulin
sensitization. Diabetes Care 2014;37:1375-1383.
27. de Rie D, Abugessaisa I, Alam T, et al. An integrated expression atlas of miRNAs and
their promoters in human and mouse. Nat Biotechnol 2017;35:872-878.
28. Abente EJ, Subramanian M, Ramachandran V, Najafi-Shoushtari SH. MicroRNAs in
obesity-associated disorders. Arch Biochem Biophys 2016;589:108-119.
29. Holscher HD. Dietary fiber and prebiotics and the gastrointestinal microbiota. Gut
Microbes 2017;8:172-184.
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