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Endocrine Research
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ierc20
Antibodies Reactive to Leptin in Adults with Type
2 Diabetes and Its Relationship with Clinical,
Metabolic and Cardiovascular Risk Parameters
Ana B. Vargas-Antillón, Mildren Porchas-Quijada, Eloy A. Zepeda-Carrillo,
Rafael Torres-Valadez, José F. Muñoz-Valle, Rafael Vázquez-Solórzano, Elia
Valdés-Miramontes, Luis A. Hernández-Palma & Zyanya Reyes-Castillo
To cite this article: Ana B. Vargas-Antillón, Mildren Porchas-Quijada, Eloy A. Zepeda-Carrillo,
Rafael Torres-Valadez, José F. Muñoz-Valle, Rafael Vázquez-Solórzano, Elia Valdés-Miramontes,
Luis A. Hernández-Palma & Zyanya Reyes-Castillo (21 Oct 2023): Antibodies Reactive to Leptin
in Adults with Type 2 Diabetes and Its Relationship with Clinical, Metabolic and Cardiovascular
Risk Parameters, Endocrine Research, DOI: 10.1080/07435800.2023.2270763
To link to this article: https://doi.org/10.1080/07435800.2023.2270763
Published online: 21 Oct 2023.
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Antibodies Reactive to Leptin in Adults with Type 2 Diabetes and Its Relationship
with Clinical, Metabolic and Cardiovascular Risk Parameters
Ana B. Vargas-Antillón
a
, Mildren Porchas-Quijada
a
, Eloy A. Zepeda-Carrillo
b,c
, Rafael Torres-Valadez
b,d
,
José F. Muñoz-Valle
e
, Rafael Vázquez-Solórzano
f
, Elia Valdés-Miramontes
a
, Luis A. Hernández-Palma
a
,
and Zyanya Reyes-Castillo
a,f
a
Instituto de Investigaciones en Comportamiento Alimentario y Nutrición, Centro Universitario del Sur, Universidad de Guadalajara, Mexico;
b
Unidad Especializada en Investigación, Desarrollo e Innovación en Medicina Genómica, Centro Nayarita de Innovación y Transferencia de
Tecnología, Universidad Autónoma de Nayarit, Mexico;
c
Hospital Civil Dr. Antonio González Guevara, Servicios de Salud de Nayarit, Mexico;
d
Unidad Académica de Salud Integral, Universidad Autónoma de Nayarit, Mexico;
e
Instituto de Investigación en Ciencias Biomédicas, Centro
Universitario de Ciencias de la Salud, Universidad de Guadalajara, Mexico;
f
Laboratorio de Biomedicina y Biotecnología para la Salud,
Departamento de Ciencias Clínicas, Centro Universitario del Sur, Universidad de Guadalajara, Mexico
ABSTRACT
Background and aims: Patients with obesity and type 2 diabetes (T2D) have shown alterations in
the anity of IgG anti-leptin antibodies which are possibly related to metabolic alterations. In the
present exploratory study, we analyzed serum samples from adults with T2D classied by body
mass index (BMI) and evaluated the relationship of IgG anti-leptin antibodies with body composi-
tion, metabolic and cardiovascular risk parameters.
Methods: Serum IgG anti-leptin antibodies (total, free and immune complexes fractions) were
measured by in-house ELISA. Body composition, metabolic biomarkers (glucose, glycated hemo-
globin, lipid prole, insulin, leptin) and cardiometabolic risk indexes (AIP, HOMA-IR, HOMA-ß) were
evaluated in one hundred T2D patients.
Results: Patients with T2D and obesity presented a decrease in the percentage of IgG anti-leptin
immune complexes compared to patients with T2D and overweight (p < 0.0053). Negative correla-
tions of IgG anti-leptin immune complexes with triglycerides (TG) (r=-0.412, p = 0.023) and VLDL-C
(r=-0.611, p = 0.017) were found in normal weight T2D patients. Free IgG anti-leptin antibodies
correlated positively with TC (r = 0.390, p = 0.032) and LDL-C (r = 0.458, p = 0.011) in overweight
individuals with T2D. Finally, total IgG anti-leptin antibodies correlated positively with leptin
hormone levels (r = 0.409, p = 0.024) and negatively with HOMA-IR (r =-0.459, p = 0.012) in T2D
patients with obesity.
Conclusions: The decrease of IgG anti-leptin immune complexes observed in patients with T2D
and obesity suggests a reduction in antibody anity to the hormone that may impact its transport
and signaling, lipid, lipoprotein and insulin metabolism.
ARTICLE HISTORY
Received June 13, 2023
Revised September 14, 2023
Accepted October 9, 2023
KEYWORDS
Anti-leptin antibodies; body
composition; leptin
resistance; metabolic
alterations; obesity
Introduction
Type 2 diabetes mellitus (T2D) is a chronic health dis-
order that represents a global health problem due to its
increasing prevalence in the last years.
1
Mexico is
among the countries with the highest diabetes preva-
lence, with an estimation of 15.7% and around
14.1 million people living with this condition.
2,3
T2D
is a multifactorial disease characterized by an increase of
glucose in blood and alterations in the metabolism of
carbohydrates, fats and proteins that are related to defi-
ciencies in the action and/or secretion of insulin.
4
Among its causes are genetic predisposition, sedentary
lifestyle, diet and obesity.
4,5
These factors promote adi-
pose tissue hypertrophy, which is a source of multiple
metabolic hormones such as leptin and other inflam-
matory mediators that promote insulin resistance (IR)
and the development of pathologies associated with
T2D, such as cardiovascular diseases.
1,5
Leptin is a key metabolic hormone implicated in
obesity and T2D. Under physiological conditions, it
acts as an anorexigenic hormone at the hypothalamus
by reducing food intake and body weight,
6
however, in
obesity and T2D, leptin resistance may contribute to the
development of metabolic disorders.
7
The mechanisms
underlying leptin resistance are not clearly understood,
but defects in hormone transport, impairment of cellu-
lar signaling, or alterations in the neuronal circuits
targeted by leptin have been suggested.
6,7
Regarding
CONTACT Zyanya Reyes-Castillo zyanya.reyes@cusur.udg.mx Instituto de Investigaciones en Comportamiento Alimentario y Nutrición, Centro
Universitario del Sur, Universidad de Guadalajara, Av. Enrique Arreola Silva No. 883, Edificio X-3, Colonia Centro, Zapotlán el Grande, Jalisco CP. 49000, Mexico
ENDOCRINE RESEARCH
https://doi.org/10.1080/07435800.2023.2270763
© 2023 Taylor & Francis Group, LLC
hormone transport, leptin was found to circulate bound
to plasma proteins, including the soluble leptin receptor
(sOB-R), C-reactive protein (CRP), as well as low-
affinity antibodies of the IgG and IgA isotype.
8,9
Recently, Bouhajja and colleagues reported that anti-
leptin antibodies may modulate the hormone biological
activity and be potentially involved in leptin resistance
in T2D.
10
In this study, they reported a decrease in the
affinity of IgG anti-leptin antibodies in T2D patients
with obesity in comparison to healthy controls, in addi-
tion, this decrease was also associated with hyperinsuli-
nemia and IR in T2D, but not in patients with obesity
and healthy controls, suggesting that the loss of anti-
body affinity to leptin may be related to disturbance of
glucose homeostasis and may favor T2D development.
10
In another study, IgG anti-leptin immunoglobulins
were analyzed in children and adolescents according to
body mass index (BMI); the fraction of antibodies
bound to leptin (immune complexes) showed
a positive correlation with BMI in children and
a negative correlation in adolescents, while the fraction
of free anti-leptin antibodies was negatively correlated
with HOMA-IR in both children and adolescents with
normal weight; suggesting that the production and affi-
nity of IgG anti-leptin antibodies can be affected by age,
body composition, and metabolic conditions.
11
In Mexican population, there is no information
regarding IgG anti-leptin antibodies in adult patients
with T2D. Analysis of serum levels of IgG anti-leptin
antibodies in these patients could add more information
to the previously suggested role of these antibodies in
the pathophysiology and metabolic alterations of T2D
patients. Therefore, the purpose of this study was to
measure the levels of IgG anti-leptin antibodies and to
evaluate its relationship with body composition para-
meters, biochemical profile, and metabolic risk indices
in patients with T2D classified by their BMI.
Materials and Methods
Patients Recruitment and Ethical Considerations
The sample size for this cross-sectional study was calcu-
lated with the formula for two means comparison
(N¼2zα þzβð Þ2S2
=d2) using a significance level of
0.05 (zα: 1.96) and desired power of 80% (zβ: 0.842), for
this formula, we considered the standard deviation (d)
of the optical density (OD) values of IgG anti-leptin
antibodies previously reported by our research
group,
11
obtaining a result of 33 individuals. One hun-
dred patients with T2D were included in this study and
were diagnosed according to the American Diabetes
Association (ADA) criteria
12
and Mexican NOM15-
SSA2-2010. Patients were recruited from the Family
Medicine Unit Number 24, “Lic. Ignacio García
Téllez” Mexican Social Security Institute, located at
Tepic, Nayarit, Mexico, in the period 2019–2020.
Sociodemographic, lifestyle and pharmacological data
were obtained through direct structured interviews
with the patient. All patients were Mexican adults
above 18 years of age (female and male), with pharma-
cological treatment with oral hypoglycemic agents and
had at least 1 year diagnosed, with laboratory-confirmed
T2D based on baseline blood glucose. Those who pre-
sented limb amputation or had a history of type 1
diabetes mellitus (T1DM), renal, thyroid, autoimmune
disease, cancer, or other self-reported chronic-disease or
with an impediment to answer the questioning (deaf or
dumb) were not included in the study.
The study was approved by the Local Health
Research and Ethic Committee 1801 (code: R-2017–
1801–13), Family Medicine Unit Number 24, Nayarit.
Mexican Social Security Institute, and was conducted
according to the principles of the Declaration of
Helsinki. The participation of all subjects was voluntary,
and the informed consent letter was signed by everyone
before their inclusion in the study.
Body Composition and Anthropometry
All body-composition and anthropometric measure-
ments were made by nutritionists. Waist circumference
was measured in the standing position, just above the
iliac crest with a stretch-resistant tape. Body fat percen-
tage (BF%) and visceral fat level were measured by
bioelectrical impedance equipment (Tanita SC-331S
body composition analyzer, Japan) following the man-
ufacturer instructions. All anthropometric measure-
ments were taken without shoes and with light
clothing. BMI was calculated by standard formula and
was used to categorize patients into 3 groups according
to the World Health Organization (WHO) criteria
13
as
follows: normal weight: BMI range 18.5 to 24.9 kg/m
2
;
overweight: BMI range 25 to 29.9 kg/m
2
; obesity: BMI
greater or equal to 30 kg/m
2
.
Biochemical Parameters, Leptin Quantication and
Metabolic Risk Indexes
Blood samples were collected after 8–12 h overnight
fasting for biochemical characterization: glucose, gly-
cated hemoglobin (HbA1c%), total cholesterol (TC),
high-density lipoprotein cholesterol (HDL-C), very low-
density lipoprotein cholesterol (VLDL-C) and triglycer-
ides (TG) were measured using standard clinical meth-
ods in the Cobas 6000 analyzer (Roche Diagnostics
2A. B. VARGAS-ANTILLÓN ET AL.
International Ltd, Risch-Rotkreuz, Switzerland), low-
density lipoprotein cholesterol (LDL-C) values were
obtained indirectly through the Friedewald formula.
14
Fasting total leptin and insulin concentrations were
assessed in serum samples using ELISA kits (Cat. No.
EIA-2395 and Cat. No. EIA-2935, respectively, both
from DRG international, Germany) according to man-
ufacturer’s instructions.
To predict the risk of a possible cardiovascular event,
the plasma atherogenic index (AIP) was estimated with
the following equation: AIP Log (TG (mg/dL)/HDL-C
(mg/dL)). Risk categories for AIP index were defined as:
low risk (AIP <0.11), intermediate risk (AIP range 0.11–
0.21) and high risk (AIP >0.21) as reported previously.
15
Evaluation of insulin resistance and pancreatic β cells
function, was performed with the formulas for homeo-
static model assessment (HOMA-IR and HOMA-B,
respectively) as follows
16
:
HOMA-IR = (Insulin (UI/mL) *Glucose(mg/dL)) / 405
HOMA-β= (20*insulin (UI/mL)) / (Glucose(mg/dL) - 3.5)
The cutoff value used for insulin resistance in adults
was > 3.80.
17
Quantication of IgG Anti-Leptin Antibodies
IgG anti-leptin antibodies were measured by an in-
house ELISA test adapted by our group
18
based on the
original protocol published by Fetissov,
19
as described
previously.
Statistical Analysis
Data distribution was verified by D’Agostino-Pearson
normality test. Variables with parametric distribution
were reported as mean ± standard deviation (SD),
while variables with non-parametric distribution
were reported as median (25–75th centiles).
Categorical variables were expressed as percentage
and absolute frequency. Differences between two
groups were assessed using Student’s t-test or Mann-
Whitney U-test according to data distribution, and
intergroup comparisons were performed using analy-
sis of variance (ANOVA) or Kruskal – Wallis followed
by Tukey’s or Dunn’s multiple comparisons post-hoc
test, according to data normality. To evaluate the
relationship between IgG anti-leptin autoantibodies
(free, total and immune complexes fractions) with
body composition and biochemical profile para-
meters, Spearman’s or Pearson’s correlation analyses
were performed, in accordance to data normality.
Analyses were carried out using GraphPad Prism
8.0.1 (GraphPad Software Inc., San Diego, CA). For
all tests, p ≤ 0.05 was considered statistically
significant.
Results
Patients Characteristics
The demographic, body composition and biochemical
characteristics of the participants with T2D are shown
in Table 1. One hundred patients were included in the
study, with a mean age of 57 ± 11 years, of which 61%
were females. Patients were classified according to
BMI and as expected, the percentage of body fat was
significantly different between the study groups, being
higher in patients with overweight (OW) or obesity
(OB), than in normal weight (NW). Visceral fat levels,
insulin and leptin levels were significantly higher in
the OB and OW groups, compared with NW. Also,
HDL-C levels were higher in the OW group in con-
trast to the NW and OB, this result may be explained
by differences in treatment, diet or physical activity
levels between the groups. The HOMA-IR values were
significantly higher in patients with OB and OW than
in NW group. The AIP value was higher in the group
with OB and NW, in contrast to patients with OW.
The values of glucose, HbA1c, TC, TG, LDL-C and
VLDL-C did not show significant differences between
the study groups. Regarding drug treatment of
patients: 93% were receiving metformin, 13% gliben-
clamide, and 17% were under insulin treatment, the
majority of these patients (76%) was in monotherapy,
whereas only 24% was receiving polytherapy with the
following combinations: metformin with insulin, met-
formin with glibenclamide or the three drugs.
Anti-Leptin Autoantibodies Analysis
IgG anti-leptin autoantibody levels in their respective
fractions (free, total and immune complexes percen-
tage) are shown in Figure 1. Free IgG anti-leptin anti-
bodies levels showed a significant difference among
groups (p < 0.0005), being higher in patients with OB
compared to patients with NW and OW (p < 0.0294).
No significant differences were observed in the fraction
of total IgG anti-leptin autoantibodies among the study
groups, while the percentage of immune complexes was
significantly lower (p < 0.0006) in patients with OB
compared to patients with OW.
ENDOCRINE RESEARCH 3
Correlation Between Anti-Leptin Autoantibodies
with Biochemical Parameters and Metabolic Risk
Indexes
We assessed the correlations between the biochem-
ical parameters with IgG anti-leptin autoantibodies
(on its free, total and immune complex fractions) in
the T2D groups. Negative correlations were observed
between TG levels (r = −0.4126, p = 0.0235) and
VLDL-C (r = −0.4077, p = 0.0253) with the percen-
tage of immune complexes in NW patients with
T2D (Figure 2a, b). Correlation analyses between
IgG anti-leptin autoantibodies with biochemical
parameters in individuals with T2D and OW show
a positive correlation with the levels of TC (r =
0.3909, p = 0.0327) and LDL-C (r = 0.4548, p =
0.0116), as seen in Figures 2c, d. Analysis of total
IgG anti-leptin autoantibodies in patients with T2D
and OB, showed a positive correlation with leptin (r
= 0.4096, p = 0.0246) and a negative correlation with
HOMA-IR (r = −0.4596, p = 0.0121) (Figure 2e, f).
Discussion
In this study, we analyzed serum levels of leptin and
anti-leptin antibodies of the IgG isotype in Mexican
patients with T2D and evaluated their relationship
with clinical, metabolic and cardiovascular risk para-
meters according to their BMI. The presence of IgG
anti-leptin autoantibodies in healthy women,
8
healthy
individuals and patients with T2D and obesity was pre-
viously reported.
10
In our study, the levels of IgG anti-
leptin autoantibodies in its free form were significantly
higher in patients with obesity compared to patients
with T2D and overweight or normal weight. Total anti-
leptin autoantibodies showed no significant differences
in T2D patients according to BMI; however, the percen-
tage of immune complexes (leptin+anti-leptin autoanti-
bodies) was significantly lower in individuals with
obesity than in overweight T2D patients. These data
are similar to the findings of Bouhajja et al.
10
; they
identified that obesity is accompanied by a decrease in
the form of leptin bound to proteins, as well as an
Table 1. Demographic characteristics, body composition, biochemical profile and metabolic risk indexes of T2D patients.
Characteristics Total cohort (n = 100)
T2D-NW
(n = 30)
T2D-OW
(n = 30)
T2D-OB
(n = 40) P value
Demographic
Age, years 57 ± 11 61 ± 11 57 ± 11 53 ± 10 -
Female, % (n) 61 (61) 50 (15) 66 (20) 65 (26) -
Alcohol intake
Does not drink, % (n) 60 (60) 60 (18) 67 (20) 55 (22) -
Social, % (n) 31 (31) 33 (10) 23 (7) 35 (14) -
Only weekends, % (n) 8 (8) 7 (2) 10 (3) 7 (3) -
Chronic, % (n) 1 (1) - - 3 (1) -
Body composition
BMI, kg/m
2
28.35 (24.63–32.65) 23.27 ± 1.23
a
27.65 (26.38–29.13)
b
33.35 (31.25–37.88)
c
<.0001
Body Fat, % 32.75 ± 8.72 24.66 ± 6.43
a
32.64 ± 5.92
b
38.89 ± 6.87
c
<.0001
Visceral fat level 10 (8–14) 8 (6–10)
a
10 ± 3
a
13 ± 3
b
<.0001
Waist circumference, cm 100 ± 13 87 ± 8
a
99 ± 6
b
108 (103–116)
c
<.0001
Biochemical profile
Glucose, mg/dL 128 (187–179) 139 (112–184) 134 (105–169) 119 (106–183) .626
Insulin, UI/mL 19.35 (10.83–26.36) 9.58 (7.94–16.56)
a
19.81 (12.49–24.63)
b
28.53 ± 13.46
b
<.0001
HbA1c, % 6.80 (5.92–8.60) 7.30 ± 1.53 6.80 (6.10–8.62) 6.90 (5.80–8.67) .787
Leptin, ng/mL 5.97 (3.73–10.12) 4.62 ± 3.34
a
6.11 (4.27–8.80)
b
9.35 ± 5.67
b
.001
TC, mg/dL 173 (157–191) 169 (153–189) 182 ± 19 165 (148–193) .147
TG, mg/dL 129 (103–180) 127 (93–189) 140 (107–177) 126 (103–185) .853
HDL-C, mg/dL 45.85 ± 11.16 43.97 ± 9.34
a
50.63 ± 11.45
b
43.68 ± 11.35
a
.017
VLDL-C, mg/dL 26 (21–36) 26 (19–37) 29 ± 11 26 (21–37) .884
LDL-C, mg/dL 98 (82–115) 89 (79–113) 102 (93–118) 99 (79–114) .260
Metabolic index
AIP, mmol/L 0.11 ± 0.25 0.12 ± 0.29 0.08 ± 0.25 0.14 ± 0.22 .571
Low risk, % (n) 39 (39) 33 (10) 43 (13) 40 (16) -
Moderate risk, % (n) 25 (25) 23 (7) 30 (9) 23 (9) -
High risk, % (n) 36 (36) 44 (13) 27 (8) 37 (15) -
HOMA-β 2.04 (0.79–3.98) 1.30 (0.76–2.50) 2.51 (1.41–4.20) 3.17 ± 3.04 .067
HOMA-IR 4.68 (2.03–9.68) 3.13 (1.86–4.45)
a
5.21 (2.57–9.51)
b
7.66 ± 6.51
b
.041
Drug treatment
Metformin, % (n) 93 (93) 90 (27) 100 (30) 90 (36) -
Glibenclamide, % (n) 13 (13) 6 (2) 16 (5) 15 (6) -
Insulin, % (n) 17 (17) 16 (5) 16 (5) 17 (7) -
Data are shown as mean ± s.d. or median (25–75th centile). NW, normal weight group; OW, overweight group; OB, obesity group; BMI, body mass index; TC,
total cholesterol; TG, triglycerides; HDL-C, high density lipoprotein cholesterol; VLDL-C, very low density lipoprotein cholesterol; LDL-C, low density
lipoprotein cholesterol; AIP, atherogenic index of plasma; HOMA-β, Homeostasis model assessment for β cell function; HOMA-IR, Homeostasis model
assessment for insulin resistance. Difference between groups was assessed by ANOVA test for parametric data and Kruskal-Wallis for non-parametric data.
P values ≤ 0.05 were considered statistically significant and are highlighted in bold.
4A. B. VARGAS-ANTILLÓN ET AL.
Figure 1. IgG anti-leptin autoantibodies levels in type 2 diabetes (T2D) patients according to BMI. IgG anti-leptin autoantibodies serum
levels are expressed in optical density (OD). A) free IgG anti-leptin autoantibodies were higher in T2D patients with OB than in OW and
ENDOCRINE RESEARCH 5
increase in plasma leptin in its free form. Likewise, they
reported that anti-leptin KD values were significantly
higher in patients with obesity, which could indicate
lower affinity of these antibodies to leptin.
10
A positive
relationship between KD and dissociation rates in
patients with obesity was identified, suggesting that
a decrease in IgG anti-leptin antibodies affinity
(evidenced by higher KD values) is associated with
hyperleptinemia, BMI, and obesity.
10
The reduced
immune complexes percentage found in our patients
with OB and T2D, are in accordance with the loss of
antibody affinity to leptin previously reported.
10
Therefore, it can be assumed that in patients with obe-
sity and T2D the affinity of antibodies to leptin is
Figure 2. Correlation analyses of IgG anti-leptin with biochemical parameters in patients with type 2 diabetes (T2D) according to BMI.
A) correlation between IgG immune complexes % and TG in T2D patients with NW. B) correlation between IgG immune complexes %
and VLDL-C in T2D patients with NW. C) correlation between free IgG anti-leptin autoantibodies with TC in T2D patients with OW. D)
correlation between free IgG anti-leptin autoantibodies with LDL-C in T2D patients with OW. E) correlation between total IgG anti-
leptin autoantibodies with leptin levels in T2D patients with OB. F) correlation between total IgG anti-leptin autoantibodies with
HOMA-IR in T2D patients with OB. R, Spearman’s or Pearson’s coefficient, as appropriate. Statistical significance was considered at p ≤
0.05.
NW groups. B) total IgG anti-leptin autoantibodies showed no significant differences according to BMI. C) IgG immune complexes
percentage was lower in OB than in OW T2D patients. Study groups: NW (n = 30), OW (n = 30), OB (n = 40). Horizontal lines indicate
mean and standard deviation. Difference between groups was assessed by ANOVA or Kruskall-Wallis followed by Tukey’s or Dunn’s
multiple comparisons post-hoc test according to data normality (*p < 0.05, **p < 0.01, ***p < 0.001). P-values ≤0.05 were considered
statistically significant.
6A. B. VARGAS-ANTILLÓN ET AL.
reduced, leading to decreased immune complexes for-
mation. Since these immune complexes have been sug-
gested to support hormone transport and stability,
8
a reduction of this antibody fraction may represent an
immunological alteration that reduces leptin availability
to initiate signaling at the hypothalamus and thereby
favor the development of leptin resistance.
Obesity is associated with a decrease in the soluble
leptin receptor, as well as a chronic inflammatory state
that may lead to leptin resistance.
20
Leptin induces the
production of pro-inflammatory cytokines such as IL-
6, which in turn promote the production of leptin,
generating a vicious cycle.
20–22
IL-6 promotes the
overexpression of inhibitory regulators of leptin sig-
naling, including SOCS3 and PTP1B, which reduce
intracellular hormone signaling.
20–22
In addition, the
inflammation associated with obesity and T2D
includes an increase in CRP.
23
It has been reported
that anti-leptin autoantibodies compete with CRP for
the binding of leptin
23
; therefore, this process may
also favor resistance to the hormone. Furthermore,
the interaction between CRP and leptin can hinder
the ability of leptin to cross the blood brain barrier
(BBB).
22
These findings indicate that, in addition to
the decrease in leptin immune complexes detected in
patients with obesity and T2D, the associated inflam-
mation (i.e., an increase in CRP
23
) may promote resis-
tance to leptin.
Although the origin of these low-affinity anti-leptin
autoantibodies remains unclear, one hypothesis involves
the phenomenon of molecular mimicry. This is
a mechanism through which infectious pathogens or
other exogenous substances, can initiate an immune
response against autoantigens due to sequence homology
with microbial peptides and cross activation of T and
B cells.
24
In specific, it has been shown that some bacterial
proteins of the intestinal microbiota have molecular
mimicry with leptin and with other appetite regulating
hormones.
9
For instance, the differences in anti-leptin
antibodies in their free, total and immune complexes
fractions may be due, in part, to the influence of the
intestinal microbiota on the production of these autoan-
tibodies. It has previously been shown that the microbiota
of patients with obesity differs from that of healthy
patients.
25,26
In addition, the use of metformin is asso-
ciated with an increase in several types of bacteria belong-
ing to the proteobacteria class, as well as Escherichia
coli.
27
Leptin shows molecular mimicry with some intest-
inal bacteria such as Lactococcus lactis, Escherichia coli,
Helicobacter pylori, and Streptococcus aglactiae.
9
Therefore, these data provide possible factors explaining
differences in production and affinity of anti-leptin anti-
bodies in T2D patients.
When evaluating the correlations of anti-leptin
autoantibodies (IgG isotype) with the biochemical
parameters of T2D patients, negative correlations
were observed between the percentage of anti-leptin
immune complexes and the concentrations of TG and
VLDL-C in patients with normal weight. It has been
reported that leptin inhibits the accumulation of lipids
in adipocytes by increasing the turnover of TG.
28
Triglycerides are exported after being packaged in
VLDL-C, the main carriers of TG in plasma.
29
In
a study carried out by Hackl et al.
29
in rat models,
they introduced intracerebroventricular leptin and
observed that optimal leptin signaling increases TG
export and decreases de novo lipogenesis in the liver.
Likewise, a chronic infusion of leptin reduced free
fatty acids, TG, ketone bodies, insulin and glucose
levels.
29
Therefore, leptin treatment stimulates the
hepatic export of VLDL-C, in addition to representing
a potential anti-steatotic mechanism of leptin action.
29
Together with these data, it could be hypothesized that
leptin immune complexes (the fraction of antibodies
bound to the hormone) favor an increase in the turn-
over of TG, by enhancing the stability and signaling of
leptin; which explains the fact that higher formation of
immune complexes is related to lower circulating
levels of TG and VLDL-C.
In T2D patients with overweight, TC and LDL-C
correlated positively with free anti-leptin antibodies.
Insulin resistance is associated with changes in lipids
and lipoprotein metabolism, as well as changes in lipo-
protein particle size that generates dyslipidemia which is
characterized by elevated VLDL-C, LDL-C and
a decrease in HDL-C.
30
In addition, insulin upregulates
LDL receptor activity by reducing LDL concentrations
under normal conditions, thus insulin resistance
appears to have a greater effect on LDL-C
metabolism.
30,31
As described previously, leptin directly
inhibits insulin secretion; therefore, leptin in rodents
and humans improves severe insulin resistance.
28,32
T2D patients with overweight of our study had higher
levels of TC and LDL-C than those with obesity and
normal weight, and this increment is correlated with
levels of free anti-leptin antibodies. Therefore,
a decrease in the formation of leptin immune complexes
can lead to a deficiency in the transport and signaling of
leptin, that indirectly affects the secretion of insulin.
Furthermore, metformin can affect the lipid metabolism
by multiple mechanisms
30
; these include changes in the
gut microbiota that are associated with reduced lipid
absorption and inhibition of bile acid absorption, with
resulting increases in LDL-C clearance or clearance
mediated by its receptor.
30,31
Considering this evidence,
metformin could have an impact on the increase in
ENDOCRINE RESEARCH 7
LDL-C in our patients, since 93% of them were under
this treatment.
In patients with T2D and obesity, a positive correla-
tion between total IgG anti-leptin autoantibodies and
leptin levels was observed, in addition, patients with
obesity had higher leptin concentration in serum, com-
pared to normal weight and overweight patients. This
last finding was somewhat expected, since concentra-
tion of leptin is highly correlated with body fat.
33
However, the relationship of anti-leptin antibodies pro-
duction with leptin concentrations may indicate an
adaptive mechanism to modulate resistance to leptin
in addition to the role of other leptin-binding proteins
previously described (including CRP
23
and soluble lep-
tin receptors).
20
Finally, the HOMA-IR was negatively correlated
with the fraction of total IgG anti-leptin antibodies
in the obesity group. This could indicate that
a greater number of total anti-leptin antibodies is
related to a decreased risk of insulin resistance, sug-
gesting a possible protective role of anti-leptin anti-
bodies for metabolic homeostasis. Of relevance, it is
known that pancreatic β-cells express leptin receptors
in their long form, so that leptin directly inhibits the
secretion of insulin and improves the severe insulin
resistance both in rodents and humans.
28,32
Furthermore, Bouhajja et al.
10
reported that KD
values of anti-leptin were positively correlated with
HOMA-IR in the obesity group, evidencing
a decreased affinity of the antibody to the hormone
that may favor T2D.
Our study has some limitations that should be
acknowledged. On one hand, patients with pre-
diabetes were not included and, therefore, it is possible
that changes in anti-leptin antibodies are also present
in these groups. It should also be considered that
timing of diagnosis and the cutoff values of HOMA-
IR may differ according to gender, ages, races and
individuals with different diseases and
complications.
34
Other factors such as poor control
of the disease, treatment schemes (monotherapy or
polytherapy) and the use of other drugs (sulfonylur-
eas) may influence HOMA-IR, metabolic and lipid
profile variables, and therefore, our results should be
interpreted cautiously.
35
On the other hand, affinity
kinetic assays are necessary in future studies to quan-
tify the precise changes in the affinity of anti-leptin
antibodies, since the values of immunocomplexes per-
centage used in this study are an indirect measure of
affinity. In addition, longitudinal studies to monitor
changes of these antibodies according to treatment, as
well as in other groups such as pre-diabetes indivi-
duals, will be of value to gain further insight of the
dynamics of these antibodies, and to explore if the
decrease of antibody affinity detected in T2D patients
with obesity, is reversible through diet and other life-
style interventions.
Conclusions
We have shown that patients with T2D and obesity
have a decrease in the percentage of anti-leptin
immune complexes of the IgG isotype, which indir-
ectly suggests a decrease in antibody affinity to leptin
that in addition to the inflammatory state associated
with obesity, may favor leptin resistance. Correlations
between anti-leptin immune complexes with TG and
VLDL-C in normal weight patients could indicate that
antibody bound to leptin favors an increase in TG
turnover, according to the hypothesis that these auto-
antibodies serve as protectors, transporters and mod-
ulators of leptin signaling. In addition, the
correlations between free anti-leptin antibodies with
TC and LDL-C in overweight individuals and T2D
could suggest a deficiency in leptin signaling that
can lead to an imbalance in insulin secretion, lipid
and lipoprotein metabolism and favor an increase in
TC and LDL-C. However, studies are lacking to verify
these results. The negative correlation of total anti-
leptin antibodies with HOMA-IR suggests a possible
protective role exerted by anti-leptin antibodies for
the homeostasis of the insulin metabolism.
Acknowledgments
This work was supported by ‘Consejo Estatal de Ciencia
y Tecnología de Jalisco (COECYTJAL), Project No. 7942,
granted to ZRC. The funding sources were not involved in
the design nor any steps of the study.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by ‘Consejo Estatal de Ciencia
y Tecnología de Jalisco (COECYTJAL), Project No. 7942,
granted to ZRC. The funding sources were not involved in
the design nor any steps of the study.
ORCID
José F. Muñoz-Valle http://orcid.org/0000-0002-2272-
9260
Rafael Vázquez-Solórzano http://orcid.org/0000-0002-
8340-7556
8A. B. VARGAS-ANTILLÓN ET AL.
Elia Valdés-Miramontes http://orcid.org/0000-0002-4544-
0291
Luis A. Hernández-Palma http://orcid.org/0000-0002-
9561-7892
Zyanya Reyes-Castillo http://orcid.org/0000-0001-6696-
0344
Authors’ Contributions
ZRC and JFMV designed the study, ABVA, MPQ, RVS,
EAZC and RTV, contributed to patient’s data acquisition
and performing experiments, ABVA and RVS analyzed
data, EVM, LAHP contributed to data interpretation.
ABVA wrote the article with contributions and suggestions
from all authors. All authors had access to the data, con-
tributed to the discussion and interpretation of the results,
and approved the final article. All authors meet the ICMJE
criteria for authorship.
Availability of Data
The datasets of this study are available on reasonable request
from the corresponding author ZRC.
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