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We performed an analytical cross-sectional study. A total of 90 individuals who met inclusion criteria were recruited, at the Instituto de Terapéutica Experimental y Clínica (INTEC) in the Centro Universitario de Ciencias de la Salud of the Universidad de Guadalajara, from 2021 to 2022.
The inclusion criteria were as follows: healthy subjects: 18–35 years old and BMI 2; obesity subjects: 18–40 years old, BMI ≥ 30 but 2, without glucose alterations; and newly diagnosed T2D subjects according to American Diabetes Association (ADA) guidelines: 30–59 years old, 25 ≤ BMI 2 [3].Exclusion criteria were as follows: hepatic failure; chronic kidney disease; diagnosis or receiving treatment for coronary, endocrine, rheumatic, and/or neoplastic disorders; acute infectious diseases; use of anti-inflammatory drugs; use of dietary supplements and/or herbal medicine; and pregnancy and breastfeeding; as well as individuals with a diagnosis for COVID-19 or presenting with compatible symptoms for a probable case for COVID-19.
Blood samples were collected from all subjects in red-capped sample tubes in the early hours of the morning with 8 h of prior fasting. The samples were then centrifuged for 10 min at 3000 rpm and the supernatant was collected and stored at −80 °C until further processing.
Weight, height, BMI, and body fat percentage were measured for each patient. BMI was calculated as weight (in kilograms) divided by the square height (in square meters), which was used as a general estimate of obesity. Body fat percentage was evaluated through body bioimpedance measurements collected using a TANITATM TBF-215 GS.
Ntn1 determination was performed by enzyme-linked immunosorbent assay from MyBioSource (San Diego, CA, USA), which recognizes natural and recombinant human-specific Ntn1, with a variation coefficient lower than 10%. hsCRP was measured similarly, using the ELISA test from MP Biomedicals (USA), diagnostics division Solon, Ohio 44139, catalog number: 07BC-1119.
The normality of data was evaluated using the Kolmogorov–Smirnoff test. The Kruskal–Wallis test was used for non-parametric results involving two or more independent samples; post-hoc analysis was performed using the Tukey test. Correlation analysis was conducted through the Spearman correlation test. Data are presented as means and standard deviations. The significance level to discard the null hypothesis was p ≤ 0.05. Statistical analyses were performed using R version 4.1.2 (R Core Team, 2021. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/, accessed on 13 January 2022). 3. ResultsThe anthropometric characteristics for all groups are shown in Table 1. Significant differences were observed between groups; for example, the mean age in the newly diagnosed T2D groups was higher than those in the healthy subjects and obesity groups (pWe observed differences in HDL (48.5 ± 7.4 vs. 44.5 ± 7.4 vs. 38.9 ± 11.2; p = 0.007), triglyceride (112.4 ± 60.2 vs. 115.6 ± 37.6 vs. 194.5 ± 78.9; p <0.001), and fasting glucose (80.2 ± 12.6 vs. 77.3 ± 11.4 vs. 118.7 ± 21.4; p < 0.001) levels between healthy, obesity, and T2D groups, respectively. The post-hoc test indicated a significant statistical difference between HDL serum concentration in healthy subjects, compared to newly diagnosed T2D (p = 0.005), while no statistical difference was found in HDL serum concentration between healthy and obesity groups, or between obesity and newly diagnosed T2D groups (p = 0.375 and p = 0.151, respectively).
Triglyceride serum concentrations differed between healthy subjects and newly diagnosed T2D groups, as well as between obesity and newly diagnosed T2D groups (p < 0.001 in both cases); however, that between healthy subjects and obesity groups did not statistically differ (p = 0.985).
No other significant difference, regarding the other cardiometabolic characteristics, was found (Table 2).The serum levels of Ntn1 significantly differed between healthy subjects and newly diagnosed T2D groups, as well as between obesity and newly diagnosed T2D groups (p < 0.001); however, there was no difference when comparing healthy subjects and obesity groups (p = 0.086). Similarly, regarding serum hsCRP, we found a statistically significant difference when we compared the three groups (p < 0.001).
The correlations between hsCRP and Ntn1, the clinical variables, and laboratory values are shown in Table 4. Ntn1 serum concentrations showed a positive correlation with hsCRP (rho = 0.443; pp = 0.05), but not with other variables. On the other hand, hsCRP serum concentrations showed a positive correlation with BMI (rho = 0.670; pp 4. DiscussionThe results of this study indicated that the serum concentration of Ntn1 is higher in subjects with a newly T2D diagnosis, compared with that in obesity and healthy subjects, suggesting an association between glucose serum concentration and inflammation status (evaluated with hsCRP) can develop at the same time.
It has been described that Ntn1 is related to the most common microvascular complications of T2D, such as diabetic retinopathy, in which angiogenesis mediated by HIF-1alpha and Vascular Endothelial Growth Factor (VEGF) is increased, as well as inflammation via NF-kB in the retina, thus explaining the pathophysiology of the disease [11,12]. In diabetic nephropathy, Ntn1 has been proposed as an early biomarker of tubular damage by inflammation with low serum concentrations of this molecule, which may lead to chronic kidney disease accompanied by loss of renal function [13,14].Other authors, such as Nevada et al., who have measured serum Ntn1 concentration in healthy, obese, pre-diabetic, and diabetic subjects, found even higher Ntn1 serum concentrations in the group composed of healthy subjects, which could be explained by the higher average age in this group, in comparison to ours (49.4 ± 12.1 vs. 22.8 ± 4.3, respectively). However, we agree on the high serum concentration of Ntn1 in individuals with T2D [15].Yim et al. have also observed a high serum concentration of Ntn1 in individuals with T2D, as well as high Ntn1 in a group composed of individuals with altered fasting blood glucose levels; however, they did not take into account the population with obesity and without insulin resistance [16].Contrary to our results, Liu et al. have found low levels of Ntn1 in individuals with T2D. They enrolled subjects who were not receiving hypoglycemic treatment at the time of Ntn1 measurement, thus differing from the present study. These differences could be explained by the characteristics of the study population, which was the main limiting factor in our study [17].Even though there is no currently defined model to explain the increased affinity of Ntn1 and its receptor (Unc5b) in individuals with T2D, we propose a possible explanation for this phenomenon: the existence and development of resistance or loss of affinity may lead to the hypersecretion of Ntn1 without negative feedback, therefore increasing the concentration of pro-inflammatory cytokines and perpetuating the inflammatory status (as measured by serum concentration of hsCRP), which will provoke an increase in NF-kB activity, thus causing more inflammation. All of these mechanisms may produce more damage to the insulin receptor, which will lead to the development of resistance and the onset of T2D and its complications. Therefore, based on the proposed inflammation pattern, we can explain the correlation between increased serum glucose, Ntn1, and hs-CRP concentrations [9,10,18].Our study has some limitations that should be considered in the interpretation of these results. First, the sample size was relatively modest. Second, being a study with a single measurement, without the follow-up of patients over time nor observation of the evolution of the behavior of Ntn1, we are planning a cohort study in which Ntn1 will be the baseline measurement and including following-up of the variables of interest, giving more weight to the results of this study.
We propose that more research aimed at the pre-diabetic population should be conducted, in order to find the exact point at which Ntn1 levels tend toward higher concentrations, thus increasing its predictive value not only for T2D complications but also for the natural progression of the disease and its prevention through timely interventions.
5. ConclusionsNtn1 levels were found to be significantly higher in individuals with newly diagnosed T2D, as well as being positively correlated with the concentrations of hsCRP and fasting blood glucose.
Ntn1 can thus be proposed as a new risk marker for the development of T2D, which would provide a new tool with which we may evaluate patients with T2D and its relationship to the inflammatory status; which, along with vascular damage, is responsible for the microvascular complications associated to T2D, as well as diabetic retinopathy and diabetic nephropathy. The aforementioned factors are the foremost causes of blindness and chronic kidney disease in our environment, invoking the need for substitutive treatment, and ultimately having direct impacts on our health system and the quality of life of patients.
Author ContributionsConceptualization, J.J.G.G. and M.G.R.-Z.; methodology, J.J.G.G. and S.P.-G.; software, J.J.G.G. and A.B.-R.; validation, M.G.R.-Z., S.P.-G. and F.G.-P.; formal analysis, M.G.R.-Z. and S.O.H.-G.; investigation, S.O.H.-G. and J.S.D.-C.; resources, D.O.S.R.; data curation, J.J.G.G. and A.B.-R.; writing—original draft preparation, J.J.G.G., M.G.R.-Z., S.P.-G., S.O.H.-G., J.S.D.-C., F.G.-P., A.B.-R. and D.O.S.R.; writing—review and editing, J.J.G.G., M.G.R.-Z., S.P.-G., S.O.H.-G., J.S.D.-C., F.G.-P., A.B.-R. and D.O.S.R.; supervision, M.G.R.-Z. and S.P.-G.; project administration, M.G.R.-Z.; funding acquisition, S.P.-G. All authors have read and agreed to the published version of the manuscript.
FundingThe development of this review was supported by parallel protocols.
Institutional Review Board StatementThis research was approved by the ethics, research, and biosafety of the Centro Universitario de Ciencias de la Salud of the Universidad de Guadalajara with the registration number CI-01621, ID CUCS/CINV/066/21, and each participant signed an informed consent upon detailed descriptions of the methods, scope, and possible results of the research.
Informed Consent StatementInformed consent was obtained from all subjects involved in the study.
Data Availability StatementThey are available to any reviewer or reader upon request. Contact the author by correspondence.
AcknowledgmentsWe thank Selene Huerta for her support and recommendations, as well as Edsaul Perez for her statistical support.
Conflicts of InterestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest.
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Figure 1. (a) Ntn1 serum concentration among study groups; and (b) hsCRP serum concentration among study groups. hsCRP, high-sensitivity C-Reactive Protein.
Table 1. Anthropometric characteristics.
Table 1. Anthropometric characteristics.
HealthyTable 2. Clinical characteristics.
Table 2. Clinical characteristics.
HealthyTable 3. Serum concentrations of Ntn1 and hsCRP.
Table 3. Serum concentrations of Ntn1 and hsCRP.
HealthyTable 4. Correlations between serum concentrations of Ntn1, hsCRP, clinical variables, and laboratory values.
Table 4. Correlations between serum concentrations of Ntn1, hsCRP, clinical variables, and laboratory values.
hsCRPNetrin 1 rhoprhopAge−0.1710.291−0.2090.196Ntn10.443<0.001------hsCRP------0.443<0.001SBP0.0930.5700.1130.489DBP 0.1720.2890.1540.343BMI0.670<0.0010.1810.263GLU−0.0700.667−0.1100.050LDL 0.1230.450−0.1120.490HDL −0.0550.7340.0640.693TG 0.1690.298−0.2580.109CT0.1750.280−0.1820.262Fat0.555<0.001−0.0210.898Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
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