Investigation of plasma leptin level in patients with type 2 diabetes mellitus

Journal of military pharmaco-medicine No7-2016

INVESTIGATION OF PLASMA LEPTIN LEVEL IN PATIENTS
WITH TYPE 2 DIABETES MELLITUS
Nguyen Van Hoan*; Nguyen Thanh Binh**; Doan Van De*; Nguyen Linh Toan***
SUMMARY
Objectives: Adipose tissue-derived hormones are associated with metabolic disorders
including type 2 diabetes mellitus (T2DM). This study investigated clinical parameters and the
levels of leptin cytokine in patients with T2DM with or without overweight/obese. Materials and
methods: Based on body mass index (BMI), 130 patients with T2DM were divided either as
overweight/obese or non-overweight/non-obese subjects. 125 overweight/obese and nonoverweight/non-obese individuals devoid of T2DM were included as healthy controls. The leptin
levels were measured in the plasma samples in all study subjects by ELISA and clinical
parameters. Results: The leptin levels were significantly lower in patients with T2DM compared
to controls ([1202.75 pg/ml median vs. 1715.4 pg/ml], p = 0.05). The leptin levels were increased
in overweight/obese T2DM patients and in overweight/obese healthy subjects compared to
without overweight/obese T2DM patients and healthy induviduals (p < 0.05). The levels of leptin
were significantly increased in female patients with T2DM and in T2DM patients with metabolic
syndrome (MS) compared to those of male patients (p < 0.001) and T2DM patients with none-MS
(p < 0.05). Conclusions: Leptin cytokine is associated with T2DM and may serve as a prognostic
marker for overweight/obese-related T2DM.
* Keywords: Type 2 diabetes mellitus; Overweight; Obesity; Leptin; Insulin.

INTRODUCTION
Type 2 diabetes mellitus is a chronic
metabolic disorder with an exponential
increase in developing countries. The
international diabetes federation (IDF)
reported 382 million diabetes cases in
2013 with a prediction of 592 million cases
by 2035, and 80% of these cases are in
developing countries [1]. In Vietnam, the

number of patients with T2DM is growing
with an estimated 3.3 million diabetes cases
reported for 2014. The prevalence of

diabetes in the age group of 30 - 69 years
is estimated to be 5.7% across Vietnam
and 7% in urban areas [2].
T2DM constitutes up to 95% of all
diabetes and is characterized by chronic
hyperglycaemia resulting from defects in
insulin secretion and/or insulin action and
metabolic disorders of protein and lipids [3].
The pathogenesis of T2DM consists of
two major abnormalities including insulin
resistance and dysfunction of insulin
production, which lead to the disability to
regulate blood glucose level [4].

* 103 Hospital
* Nghean Endocrine Hospital
*** Military Medical University
Corresponding author: Nguyen Linh Toan ()

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Journal of military pharmaco-medicine No7-2016
The damage of pancreatic β cells to
produce sufficient insulin and adiponectin
as well as an increased production of

pro-inflammatory cytokines due to obesity
are the major contributing factors for T2DM
[3]. Insulin resistance appeared years before
the clinical manifestation of T2DM and
is significantly associated with obesity,
especially with abdominal and visceral
obesity with an abnormally increased
waist-to-hip ratio (WHR), dyslipidemia,
hypertension and other metabolic disorders.
Therefore, obesity largely contributes to
insulin resistance in patients with T2DM [5].
Adipose tissue is recently recognized
as an organ for metabolism of sexual
steroids and production of adipsin that
significantly contributes to loss of body
weight [6]. Adipose tissue-derived proteins
regulate metabolic functionalities including
hormone activities [7]. Adipose tissue is
also known as an endocrine organ that can
produce various peptides with bioactivities
namely adipokines. Increased adipose tissue
due to obesity, especially deposition of
visceral fat, is associated with insulin
resistance, increased blood glucose levels,
lipid metabolic disorders, hypertension
and inflammation [7]. The adipose tissuederived hormones such as adiponectin,
leptin, adipsin, and resistin have been
shown to be associated with metabolic
disorders and are risk factors for T2DM
and cardiovascular diseases. This study

aims to investigate the levels of leptin and
their correlations with insulin resistance of
overweight/obese and T2DM.
42

MATERIALS AND METHODS
1. Patients and controls.
One hundred thirty (n = 130) patients
with T2DM and one hundred twenty five
(n = 125) control individuals were included
in the study. The patients were classified
into two subgroups based on their BMI and
T2DM status. The first subgroup includes
the patients with both overweight/obese
(BMI ≥ 23) and T2DM (patients with
overweight T2DM, n = 57). The second
subgroup includes patients with T2DM
but without overweight (BMI < 23) (nonoverweight T2DM, n = 73). The patients
were diagnosed for T2DM based on
the standard criteria reported by world
health organization (WHO) in 1998 and by
International Diabetes Federation (IDF) in
2005. The anthropometric indicators such as
height, weight, waist and hip circumference
were measured for all study participants.
BMI and waist-hip ratio (WHR) were
calculated based on their anthropometric
indicators (table 1).
All the individuals in the control group
were clinically examined and were considered

healthy during sampling. None of them
had any chronic infectious diseases or
conditions such as hepatitis, liver cirrhosis,
obstructive pulmonary disease, gout and/or
any infection. The control group was also
further divided into two subgroups. The
first control subgroup include normal healthy
individuals, (non-overweight control individuals,
n = 57), with fasting venous blood glucose
test < 5.6 mmol/L, blood pressure < 130
mmHg and < 85 mmHg, ECG in normal
limits, other tests in the normal range,


Journal of military pharmaco-medicine No7-2016
BMI from 18.5 to 23. The second control
subgroup is healthy individuals with BMI
≥ 23 (overweight/obese control individuals,
n = 68), fasting venous blood glucose test
< 5.6 mmol/L, blood pressure < 130 mmHg,
and < 85 mmHg, ECG in normal limits
(table 1).
2. Measurement of biochemical
parameters.
The levels of lipid components including
cholesterol (CT), triglycerides (TG), highdensity lipoprotein - cholesterol (HDL-C),
low-density lipoprotein - cholesterol (LDL-C)
were measured by using automatic
biochemical AVADIA 1800 (Siemens, USA).
Fasting glucose levels were measured in

blood by measurement of the UV with
Hexokinase. Insulin levels were measured
by the Achitech i2000SR (Abbott, USA).
Blood fasting glycosylated hemoglobin
(HbA1c) levels were quantified by the ionexchange method using high performance
liquid chromatography (HPLC). Insulin
resistance index was evaluated according
to the homeostasis model assessment
insulin resistance index (HOMA-IR index)
and the quantitative insulin sensitivity check

insulin resistance index (QUICK-IR index).
In addition, the β-cell function (homeostatic
model assessment- HOMA-β) and the
insulin secretion of β-cell were evaluated
according to Matthews’ method.
3. Measurement of leptin levels.
The levels of leptin were measured in
the respective plasma samples of the study
participants by using a commercial ELISA
kit following the manufacturer’s instruction
(Human leptin, Sigma, USA).
* Statistical analysis:
Clinical and demographic data were
presented in median values with range for
continuous variables. The student’s t-tests
or one-way ANOVA were used for comparing
mean of two or more groups, respectively.
Chi-square or Fisher’s exact tests were
used to compare categorical variables.

Kruskal-Wallis or Mann-Whitney U test was
used to analyze the plasma levels of
leptin in the patients with T2DM and in
controls wherever appropriate. All statistical
analyses were performed using IBM
Statistics SPSS v.19 (IBM Corp, Armonk,
NY. the USA), and the level of significance
was set at a P value of less than 0.05.

RESULTS
1. Demographic, clinical and biochemical characteristics of the study subjects.
Table 1: Characteristics of patients with type 2 diabetes mellitus and controls.
Type 2 diabetes mellitus
Characteristics

Age (years)
Gender (M/F)

Without type 2 diabetes mellitus

NonOverweight/obese
overweight p value
T2DM, n = 73
T2DM, n = 57

p value (*)

Overweight/obese Non-overweight
individuals,
individuals,

p value
n = 68
n = 57

57.82 ± 7.98

58.89 ± 6.99

NS

55.88 ± 8.54

57.84 ± 6.93

NS

< 0.05

46/27

32/25

NS

28/40

13/44

0.02

0.03

BMI

26.03 ± 2.24

21.24 ± 1.37 < 0.0001

24.99 ± 1.7

20.69 ± 1.63

< 0.0001

NS

WHR

0.93 ± 0.04

0.92 ± 0.07

0.91 ± 0.08

0.88 ± 0.06

< 0.05

0.001

< 0.05

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Journal of military pharmaco-medicine No7-2016
Fasting
glucose
(mmol/L)

9.50 ± 3.07

8.63 ± 2.07

NS

5.19 ± 0.34

5.17 ± 0.35

NS

< 0.0001

Total Cholesterol
(mmol/L)

5.14 ± 0.99

5.17 ± 0.75

NS

5.13 ± 0.97

5.09 ± 1.02

NS

NS

Triglyceride (mmol/L)

2.91 ± 2.65

2.22 ± 1.59

NS

2.08 ± 2.10

1.59 ± 1.13

NS

< 0.05

HDL-C (mmol/L)

1.15 ± 0.25

1.27 ± 0.52

NS

1.31 ± 0.32

1.45 ± 0.37

NS

NS

LDL-C (mmol/L)

2.70 ± 0.82

2.93 ± 0.74

NS

2.95 ± 0.88

2.99 ± 1.06

NS

NS

HbA1c (%)

7.37 ± 1.23

7.24 ± 1.3

NS

5.63 ± 0.57

5.6 ± 0.28

NS

NS

Insulin (pmol/L)

10.64 ± 9.87

7.30 ± 4.30

< 0.001

7.33 ± 2.42

6.32 ± 2.94

NS

< 0.0001

HOMA-RI

4.90 ± 6.15

2.82 ± 1.77

< 0.05

1.69 ± 0.58

1.46 ± 0.70

< 0.05

< 0.0001

QUICKI

0.81 ± 0.10

0.85 ± 0.08

< 0.01

0.92 ± 0.07

0.97 ± 0.10

< 0.01

< 0.0001

HOMA-β

38.80 ± 35.83

31.27 ± 20.78

> 0.05

92.12 ± 47.35

78.60 ± 38.79

> 0.05

< 0.0001

(*) Comparison between type 2 diabetes mellitus and non-diabetes mellitus.
The mean age of the patients with T2DM was higher than control individuals
(without T2DM) (p < 0.05), while there was no difference in mean age between the
patients with overweight T2DM and non-overweight T2DM. The proportion of male
patients with T2DM was higher than the control group (p = 0.03).
The levels of fasting glucose, triglycerides were significantly higher in the patients with
T2DM compared to control individuals (p < 0.05). The levels of insulin and homeostasis
model assessment insulin resistance (HOMA-RI) were significantly higher in the patients
with T2DM compared to control individuals (p < 0.001). In contrast, the levels of quantitative
insulin sensitivity check index (QUICKI) and homeostatic model assessment β-cell function
(HOMA-β) were significantly lower in the patients with T2DM compared to control individuals

(p < 0.001).
2. Leptin levels in patients with type 2 diabetes mellitus and in controls.

Figure 1: Leptin level in type 2 diabetes mellitus (T2D) patients and heathy controls (HC).
(A) Leptin level was measured in the plasma of patients with T2DM and healthy controls
and was compared between groups. (B) Leptin level in patients with overweight/obese
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Journal of military pharmaco-medicine No7-2016
(OV/O) T2DM and those with-without overweight/obese (WOV/O) T2DM and in OV/O
and WOV/O control individuals. P values were calculated by using Mann-Whitney U test.
The leptin levels were significantly lower in the patients with T2DM [median: 1202.75
pg/ml] compared to the controls [median: 1715.4 pg/ml] (p = 0.05) (figure 1A). Among
the patients with T2DM, the leptin levels were significantly lower in the T2DM patients
with WOV/O compared to those with OV/O T2DM (p < 0.05). Among the control group,
the leptin levels were significantly increased in the overweight/obese individuals compared
to the WOV/O individuals (p < 0.05) (figure 1B).

Figure 2: Leptin level in male and female patients with T2DM, in the T2DM patients
with and without MS and in overweight/obese levels of subjects.
The levels of leptin were significantly increased in female patients with T2DM and in
T2DM patients with MS compared to those of male patients (p < 0.001) and T2DM patients
with none-MS (p < 0.05) (figure 2A and 2B). The levels of leptin were significantly increased
associated to BMI of subjects (p < 0.001) (figure 2C).
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Journal of military pharmaco-medicine No7-2016
DISCUSSION

Leptin, a hormone secreted by adipocytes
in quantities which mainly reflect fat mass
and serves as an important signal of body
energy stores. Leptin deficiency in mice
and/or in humans is associated with
neuroendocrine and metabolic abnormalities,
including insulin resistance and diabetes.
All these abnormalities are corrected by
exogenous leptin administration, suggesting
that leptin plays a role in glucose homeostasis and possibly in the pathogenesis of
other obesity-related metabolic complications
[4].
Previous studies have shown that plasma
leptin levels are closely associated with
adiposity [8], and its level correlated with
increased MS components [9]. Plasma leptin
level in diabetes is still controversial;
one recent study does not observe any
significant difference between diabetic and
non-diabetic subjects, others reported
significant lower level in type 1 diabetic
subjects and type 2 diabetics with similar
adiposity [10]. In line with other studies,
our results revealed that leptin levels were
significantly increased in OV/O patients
with T2DM and in OV/O healthy individuals
compared to patients with WOV/O T2DM
and healthy individuals, respectively
(figure 1). In addition, our investigations
revealed that leptin levels were increased

in female T2DM patients and in T2MS
patients with MS. The increasing leptin
levels were associated with BMI of
subjects (figure 2). These results indicate
that increased leptin may potentially respond
to the development of overweight/obese and
T2DM and metabolic disorders.
46

Recently studies shown that low leptin
levels were also associated with the
pathogenesis of many other diseases
such as lipid metabolic disorders and
obesity. The leptin level was significant
correlated to T2DM clinical parameters
including insulin (r = 0.23, p < 0.05) and
HOMA-β (r = 0.216, p < 0.05) and inversity
correlated to QUICKI (r = -0.366, p < 0.0001).
However, our data show that the leptin
levels are not correlated with numerous
clinical parameters such as triglycerides,
cholesterol, HDL-C and LDL-C (data not
shown). Those observations indicate that
adiponectin levels involve in the development
of T2DM. Therefore, based on the levels
of leptin, an adipose-derive cytokine could
be useful for diagnosis of insulin resistance.
However, more studies are required to
propose a new index and verify the
diagnostic accuracy in clinical practice

and to establish a cut-off value and reference
range of insulin sensitivity for specific
populations [11].
CONCLUSON
Our study demonstrated that the levels
of leptin are significantly modulated during
the development of overweight/obese and
T2DM. The leptin levels were significantly
associated to BMI, genders and MS and
with clinical parameters of obesity and
T2DM. Leptin may possibly modulate the
pathogenesis of overweight and T2DM.
Acknowledgements
We thank all the study subjects for
their participation. This research is funded
by Vietnam National Foundation for Science
and Technology Development (NAFOSTED)
under grant number 106-YS.02-2014.36.


Journal of military pharmaco-medicine No7-2016
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