Key words insulin resistance - type 2 diabetes mellitus - nesfatin-1
Introduction
Nesfatin-1, derived from the nucleobindin2 (NUCB2)precursor, contains 82 amino acids
and is highly conserved in humans, rats and mice. It has been reported that nesfatin-1
suppresses nocturnal food intake and reduces body weight gain when injected into the
third ventricle of rodents, whereas infusion of NUCB2 antisense oligonucleotide stimulates
food intake ([Oh-I S et al., 2006 ]; [Johnstone et al., 2006 ]; [Stengel et al., 2009 ]). Furthermore, recent studies have shown that nesfatin-1 reduces food intake in
rodents when administered peripherally ([Shimizu et al., 2009 ]). Plasma levels of nesfatin-1 are reduced by fasting, and increased after refeeding
([Kohno et al., 2008 ]). This suggests that nesfatin-1 plays a role in satiety regulation and, possibly,
energy homeostasis ([Kohno et al., 2008 ]).
The nesfatin-1 is prominently expressed in several regions of the hypothalamus and
spinal cord autonomic nuclei. Also, it has been demonstrated to be present in peripheral
tissues including adipocytes, gastric mucosa and in human and rat pancreatic beta-cells,
indicating the possible involvement of nesfatin-1 in the regulation of insulin secretion
from pancreatic beta-cells ([Stengel et al., 2009 ]; [Goebel et al., 2009 ]; [Foo et al., 2010 ]).
Obesity is mainly caused by excessive food intake and/or reduced energy expenditure.
It is an important public health problem and is associated with insulin resistance
and T2DM ([Rasouli and Kern, 2008 ]). As nesfatin-1 may play an important role in the regulation of body weight and
insulin secretion, we hypothesized that plasma nesfatin-1 levels might be affected
by insulin resistance in subjects with newly diagnosed type 2 diabetes mellitus (nT2DM)
and impaired glucose tolerance (IGT). We, therefore, measured plasma nesfatin-1 levels
in these groups and correlated them with anthropometric and metabolic parameters.
Materials and Methods
Study patients
220 Chinese volunteers were involved in this study and categorized into 3 groups.
74 patients with nT2MD (39 men and 35 women; age 54±11 years; body mass index (BMI),
25.0±3.7 kg/m2 , nT2MD group) and 73 subjects with IGT (35 men and 38 women; age 54±10 years; BMI,
24.7±2.7 kg/m2 , IGT group) were recruited in the study. T2DM and IGT were diagnosed with a 75 g
oral glucose tolerance test (OGTT) according to Word Health Organization criteria
([Alberti and Zimmet, 1998 ]). The patients with IGT and T2DM were newly diagnosed and had not previously received
any diabetic medications or diets. 73 age-and BMI-matched healthy subjects who had
normal glucose tolerance (36 men and 37 women; age 51±7 years; BMI, 24.5±3.6 kg/m2 , NGT group) were selected as controls. None of healthy subjects had a family history
of diabetes or other endocrine disorders or were taking any medication known to alter
glucose tolerance. Their body weight was stable for at least 2 months. Patients with
type 1 diabetes mellitus, acute or chronic infectious disease, pregnancy, heart failure,
hypertension, liver or kidney disease and cancer were excluded from the study. All
study subjects were of Han Chinese origin. This study was approved by the Ethics Committee
of Chongqing Medical University, and informed consent was obtained from all study
participants.
Anthropometry and blood samples
Anthropometric measurement was performed in the morning, before breakfast, with subject
wearing light clothing, without shoes. Body weight and height were measured in all
subjects using a scale and a wall-mounted stadiometer to the nearest 0.5 kg and 0.5 cm
respectively. Waist and hip circumferences were measured using standard techniques.
The waist to hip ratio (WHR) was calculated as the ratio of waist and hip circumferences.
A 75 g oral glucose tolerance test was conducted after a 12 h overnight fast. Body
mass index (BMI)was calculated as the body weight in kilograms divided by the height
in meters squared. The homeostasis model assessment of insulin resistance (HOMA-IR) and insulin secretion (HOMA-IS) were calculated using the following equations ([Albareda et al., 2000 ]): HOMA-IR =fasting insulin (μU/ml)×fasting glucose (mmol/L)/22.5, and HOMA-IS =[20×fasting insulin (μU/ml)]/[fasting blood glucose (mmol/L) – 3.5].
Venous blood samples were obtained at 0, 30, 60, 120 min after glucose load. Plasma
glucose and glycosylated hemoglobin (HbA1c) were soon measured by the glucose-oxidase method and anion exchange HPLC respectively.
Plasma samples were frozen and stored at − 70°C for the measurements of nesfatin-1,
insulin, free fatty acid (FFA) and blood fat levels.
Analytical procedure
Plasma insulin was measured by radioimmunoassay (RIA) using human insulin as standard
(Institute of atomic energy, China). FFA was measured with a commercial kit (Randox
Laboratories Ltd, Antrim, UK). Samples for measurement of plasma levels of total cholesterol
(TC), high-density lipoprotein cholesterol (HDL-C)and triglycerides (TG) were drawn
in lithium-heparin vacuum tubes and analysed enzymatically using an autoanalyzer (Hitachi
747; Hitachi, Tokyo, Japan). Low-density lipoprotein cholesterol (LDL-C) was calculated
using the Friedewald equation (LDL-C (mmol/l)=total cholesterol – HDL-C – TG/2.2)
[(Friedewald et al., 1972) ].
Plasma nesfatin-1 assay
Plasma nesfatin-1 levels were measured using a commercially available enzyme-linked
immunosorbent assay (ELISA, Phoenix Pharmaceuticals, Belmont, Calif). The linear range
of the assay was 0.78–50 µg/L. The inter-assay and the intra-assay coefficients of
variation were 10% and 15%, respectively.
Statistical analysis
Statistical analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago,
IL, USA ), and P <0.05 (two-tailed) was considered statistically significant. Data was shown as the
mean±SD. We used one-way ANOVA with post hoc (least significant difference) analysis
to assess for differences in body composition, anthropometric, metabolic, and hormonal
parameters among the T2DM, IGT, and normal controls. Simple and multiple linear regression
analyses were used to examine the association between fasting nesfatin-1 levels and
other biomarkers. The associations of nesfatin-1 with IGT and diabetes were examined
by multivariate logistic regression analysis that contains: 1) nesfatin-1, age and
gender; 2) nesfatin-1, age, gender, BMI and WHR; 3) nesfatin-1, age, gender, BMI,
WHR and lipid profile.
Results
The clinical characteristics and biochemical results of the 3 groups were given in
[Table 1 ]. Patients with nT2DM and IGT had increased levels of fasting blood glucose (FBG),
2 h blood glucose after a 75 g glucose load (2hPBG), 2 h plasma insulin after the
glucose load (2hINS), HbA1C , HOMA-IR , and decreased levels of HOMA-IS , when compared with the controls (P <0.05 or P <0.01 [Table 1 ]). The WHR and TG in nT2DM patients were also significantly higher than those in
the control subjects. Plasma nesfatin-1 levels were significantly increased in both
the nT2DM and IGT groups when compared with the controls (1.91±0.79 and 1.80±0.80
vs. 1.41±0.58 μ g/L, both P <0.01, [Fig.1 ]), but there were no significant differences between the nT2DM and IGT groups. After
adjusted for differences in BMI, plasma levels of nesfatin-1 were still higher in
the nT2DM and IGT groups than the controls (1.91±0.10 and 1.80±0.10 vs. 1.40±0.10 μg/L,
both P <0.01). Other parameters, such as BMI, fasting plasma insulin (FINS), TC, HDL-C, LDL-C,
FFA, did not differ among the different groups. There were no age and gender differences.
Fig. 1 Plasma nesfatin-1 levels in 3 groups nT2DM, newly type 2 diabetes mellitus; IGT,
impaired glucose tolerance; NGT: normal glucose tolerance vs. NGT group **
P <0.01.
Table 1 Clinical characteristic of study groups (x ±s ).
Group
nT2DM
IGT
NGT
* Values are mean±SD or frequency (percent). nT2DM, newly diagnosed type 2 diabetes
mellitus; IGT, impaired glucose regulate; NGT, normal glucose tolerance; BMI, body
mass index; FBG, fasting blood glucose; 2 h-PBG, 2 h blood glucose after a 75 g glucose
load; FFA, free fatty acids; FINS, fasting plasma insulin; 2 hINS, 2 h plasma insulin
after a glucose load; HOMA-IR , HOMA-insulin resistance index; HOMA-IS , HOMA-β cell secretion index; HDL-C, High-density lipoprotein cholesterol; LDL-C,
Low-density lipoprotein cholesterol. *
**
†
‡
n (male/female)
74 (39/35)
73 (35/38)
73 (36/37)
age
54±11
54±10
51±7
BMI (kg/m2 )
25.0±3.7
24.7±2.7
24.5±3.6
waist hip ratio
0.91±0.05*
0.90±0.08
0.87±0.08
FBG (mmol)
10.9±3.8**‡
6.0±0.4**
5.4±0.4
2h-PBG (mmol/L)
21.2±9.7**‡
8.9±1.3**
5.93±0.81
FINS (mU/L)
8.26±4.25
8.82±3.05
7.74±2.85
2h-INS (mU/L)
45.89±38.52*†
60.48±36.97**
26.37±16.72
HOMA-IR
3.79±1.77**‡
2.37±1.06**
1.85±0.66
HOMA-IS
31.0±28.0**‡
72.2±35.2**
88.8±49.4
Hb A1 c (%)
8.8±2.4**‡
5.9±0.4**
5.5±0.4
triglyceride (mmol/L)
2.08±1.56**
1.63±1.02
1.34±0.68
total cholesterol (mmol/L)
5.25±1.4
4.98±1.33
4.83±0.83
HDL-C (mmol/L)
1.29±0.69
1.22±0.37
1.27±0.29
LDL-C (mmol/L)
3.01±1.09
2.73±0.81
2.69±0.68
FFA (mmol/L)
0.68±0.31
0.73±0.51
0.66±0.47
We used linear regression analysis to examine the association between plasma nesfatin-1
levels and other metabolic parameters related to insulin resistance. Plasma nesfatin-1
was positively correlated with BMI ([Fig. 2 ]), HbA1C , FBG, 2hPBG, FINS and HOMA-IR with simple regression analysis using the pooled data ([Table 2 ]). After adjustment for BMI, plasma nesfatin-1 remained positively correlated with
HbA1C (r =0.21, P <0.01), FBG (r=0.24, P <0.01), 2hPBG (r =0.27, P <0.01), FINS (r =0.16, P <0.05) and HOMA-IR (r =0.28, P <0.01). On the other hand, with multiple stepwise regression analysis, only HOMA-IR and BMI remained independently correlated with plasma nesfatin-1 levels ([Table 2 ]). The multiple regression equation was: (Y=0.142XHOMA-IR + 0.043XBMI + 0.269). Multivariate logistic regression analysis showed that plasma nesfatin-1
was significantly associated with IGT and T2DM even after controlling for anthropometric
variables and lipid profile ([Table 3 ]).
Table 2 The results of linear regression analysis of variables associated with plasma nesfatin-1
levels in study subjects.
Variable
Simple
Multiple
Estimate
P-value
Estimate
P-value
95%CIs
2h-PBG, 2h blood glucose after a 75 g glucose load; 2hINS, 2h plasma insulin after
the glucose load; HOMAIR , HOMA-insulin resistance; HOMA-IS, HOMA-β cell secretion index; HDL-C, high-density
lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol
age
0.62
0.434
–
–
body mass index
0.043
0.013
0.063–0.213
waist-to-hip ratio
0.044
0.581
–
–
triglyceride
0.141
0.076
–
–
total cholesterol
0.008
0.920
–
–
HDL
0.099
0.214
–
–
LDL
− 0.060
0.453
–
–
free fatty acids
0.032
0.689
–
–
hemoglobin A1 c
0.177
0.025
–
–
FBG
0.206
0.009
–
2h-PBG
0.248
0.002
–
–
fasting plasma insulin
0.213
0.007
–
–
2hINS
0.103
0.201
–
–
HOMAIR
0.309
0.000
0.142
0.000
0.011–0.077
HOMAIS
− 0.036
0.654
–
–
Table 3 Association of plasma nesfatin-1 with IGT and Type 2 diabetes in fully adjusted models.
Model adjusted for
IGT
nT2DM
OR
95%CI
P-value
OR
95%CI
P-value
nT2DM, newly type 2 diabetes mellitus; IGT, impaired glucose tolerance; NGT: normal
glucose tolerance; BMI, body mass index; WHR, waist hip ratio; TG, triglyceride; TC,
total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density
lipoprotein cholesterol; FFA, free fatty acids; OR: odds ratio
age, gender
0.037
0.004–0.326
0.003
0.052
0.006–0.442
0.007
age, gender, BMI, WHR
0.023
0.002–0.220
0.001
0.003
0.004–0.309
0.003
age, gender, BMI, WHR TG, TC, HDL-C, LDL-C, FFA
0.019
0.002–0.199
0.001
0.032
0.003–0.331
0.004
Fig. 2 Correlations of plasma nesfatin-1 levels with BMI in study subjects r =0.234, P <0.05.
Discussion
In the present study, we demonstrated that when compared to control subjects, plasma
nesfatin-1 levels were elevated both in patients with nT2DM and with IGT. These results
were consistent with a similar observation showing that circulating and adipose tissue
protein levels of nesfatin-1 were higher in diet-induced obese mice ([Ramanjaneya et al., 2010 ]). Type 2 diabetes mellitus is a genetically heterogeneous disorder and is associated
with insulin resistance and impaired insulin secretion ([Taylor et al., 1994 ]). Its incidence is rising, in parallel with the growing epidemic of obesity. Nesfatin-1
is a newly discovered physiological regulator of food intake. When injected into the
third brain ventricle, nesfatin-1 significantly decreased food intake and body weight
in rats ([Oh-I et al., 2006 ]; [Johnstone et al., 2006 ]; [Stengel et al., 2009 ]). The physiological consequences of elevated plasma nesfatin-1 in patients with
nT2DM or IGT found in present study are uncertain. Via its putative anoretic action,
it may result in reduced body fat, and improved insulin sensitivity. However, the
roles of nesfatin-1 in the pathogenesis of insulin resistance and T2DM are presently
not well understood.
The another report on plasma nesfatin-1 levels in humans showed that it was lower
in T2DM patients compared with controls ([Li et al., 2010 ]). Differences in study design, including patient selection (e. g., obese vs. lean,
glycemia level, diet type)and experimental conditions, may have contributed to the
discrepancy between these results and ours. For instance, Li QC et al. ([Li et al., 2010 ]) studied patients at various stages of the disease, some of whom had macrovascular
and microvascular complications and were on multiple medications including oral hypoglycemic
drugs and insulin, all of which could affect the release of nesfatin-1 ([Gonzalez et al., 2009 ]). These may thus be limiting the power of the analysis. In comparison, in the present
study, patients were newly diagnosed, were not treated with oral hypoglycemic agents,
nor were on special diets. A previous study in vitro had shown that the release of
NUCB2/nesfatin-1 from isolated rat islets significantly increased in response to glucose
([Foo et al., 2010 ]). Thus it is possible that β-cells secrete normal amounts of nesfatin-1 and insulin
only during the early stages of diabetes. Hence, we designed this study based on excluding
the effects of macrovascular and microvascular complications and multiple medications
on plasma nesfatin-1.
With simple regression analysis, plasma nesfatin-1 levels were significantly correlated
with BMI, which was in agreement with recent studies ([Ramanjaneya et al., 2010 ]; [Tan et al., 2011 ]; [Ogiso et al., 2011 ]), but in disagreement with another study ([Aydin et al., 2009 ]). We studied patients with nT2DM, who differed from the studies mentioned above.
As a novel adipokine, nesfatin-1 is increased in obese states in both rodents and
humans ([Ramanjaneya et al., 2010 ]). For instance, Tan et al. studied subjects consisted of Caucasian (14 overweight,
BMI, 25.0 to < 30 kg/m2 ; 10 obese, BMI, ≥ 30kg/m2 ), who differed from our studies with respect to BMI (74 nT2MD, BMI, 25.0±3.7 kg/m2 ; 73 IGT, BMI, 24.7±2.7 kg/m2 ) ([Tan et al., 2011 ]). Thus, ethnicity and patient selection (e. g., Caucasian vs. Chinese; obese vs.
lean; glycemia level) may explain the different levels of nesfatin-1 reported by Tan
et al. ([Tan et al., 2011 ]). In addition, the differences of study designs and experimental conditions likely
contribute to the disparity. However, as with most new discoveries, these findings
need to be reproduced.
Simple regression analysis demonstrated that plasma nesfatin-1 level was also positively
correlated with HbA1C , FBG, 2hPBG, FINS and HOMAIR . Usually, increased plasma levels of HbA1C , FBG, 2hPBG and FINS were caused by insulin resistance, and aggravated T2DM. In multiple
stepwise regression analysis, only HOMA-IR and BMI were independently related factors with plasma nesfatin-1 levels. Therefore,
a possible reason for the increased levels of nesfatin-1 in nT2DM patients might be
insulin resistance, as has been suggested by a previous study ([Ramanjaneya et al., 2010 ]).
Our study was limited by its cross-sectional design, a relatively small sample size
and therefore did not prove a causal relationship between increased plasma nesfatin-1
levels and the development of T2DM.
In conclusion, the novelty of our study is that we demonstrate, for the first time,
elevated nesfatin-1 levels in nT2DM and IGT. Furthermore, plasma nesfatin-1 levels
are associated with several metabolic and anthropometric parameters in nT2DM and IGT
subjects. Association with these metabolic indices suggests that elevated nesfatin-1
levels may be an anorexigenic factor that opposes weight gain and improves insulin
resistance.
