PLOS ONE RESEARCH ARTICLE FGF21 serum levels are related to insulin resistance, metabolic changes and obesity in Mexican people living with HIV (PLWH) Arguiñe Ivonne Urraza-Robledo1,2, Marta Giralt3, Faviel Francisco González-Galarza4, Francesc Villarroya3, Alberto Alejandro Miranda Pérez5, Pablo Ruiz Flores4, Marı́a Elena Gutiérrez Pérez5, Peré Domingo6, Francisco Carlos López-Márquez ID4* 1 Center for Biomedical Research, Medicine School Autonomous University of Coahuila (IMSS), Torreón, Mexico, 2 High Specialty Medical Unit (UMAE) # 71, Mexican Social Security Institute, Ciudad de México, a1111111111 Mexico, 3 Departament de Bioquı́mica i Biomedicina Molecular and Institut de Biomedicina (IBUB), CIBER a1111111111 Fisiopatologı́a de la Obesidad y Nutrición, Universitat de Barcelona, Barcelona, Spain, 4 Center for a1111111111 Biomedical Research, Faculty of Medicine, Autonomous University of Coahuila, Torreón, Mexico, a1111111111 5 Biomedical Research Center Medicine School Autonomous University of Coahuila, Torreón, Mexico, a1111111111 6 Department of Infectious Diseases, Hospital de la Santa Creu i Sant Pau, Institut de Recerca del Hospital de la Santa Creu i Sant Pau, Barcelona, Spain * francisco.lopez@uadec.edu.mx OPEN ACCESS Citation: Urraza-Robledo AI, Giralt M, González- Abstract Galarza FF, Villarroya F, Miranda Pérez AA, Ruiz Flores P, et al. (2021) FGF21 serum levels are Background related to insulin resistance, metabolic changes Antiretroviral therapy has significantly improved prognosis in treatment against HIV infec- and obesity in Mexican people living with HIV (PLWH). PLoS ONE 16(5): e0252144. https://doi. tion, however, prolonged exposure is associated to cardiovascular diseases, lipodystrophy, org/10.1371/journal.pone.0252144 type 2 diabetes, insulin resistance, metabolic alteration, as obesity which includes the accu- Editor: Ying-Mei Feng, Capital Medical University, mulation of oxidative stress in adipose tissue. FGF21 is a peptide hormone that is known to CHINA regulate glucose and lipid metabolism. FGF21 is expressed and secreted primarily in the Received: June 15, 2020 liver and adipose tissue, promoting oxidation of glucose/fatty acids and insulin sensitivity. Alterations in FGF21 may be associated with the development of insulin resistance, meta- Accepted: May 11, 2021 bolic syndrome and cardiovascular disease. We hypothesized that FGF21 protein levels are Published: May 21, 2021 associated with metabolic abnormalities, placing special attention to the alterations in rela- Copyright: © 2021 Urraza-Robledo et al. This is an tion to the concurrence of overweight/obesity in people living with HIV (PLWH). open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and Design reproduction in any medium, provided the original Serum FGF21 was analyzed in 241 subjects, 160 PLWH and 81 unrelated HIV-uninfected author and source are credited. subjects as a control group. Clinical records were consulted to obtain CD4+ cell counting Data Availability Statement: All relevant data are and number of viral RNA copies. Serum FGF21 levels were tested for correlation with within the manuscript. anthropometric and metabolic parameters; glucose, cholesterol, HDL, LDL, VLDL, triglycer- Funding: We received funding from The State ides, insulin and indexes of atherogenesis and insulin resistance (HOMA). Council of Science and Technology (FONCYT) COAH-2019-C13-C054, for the purchase of reagents used in this project, in addition to having a Results grant from supported by PI17/00498 & PI17/ The participants were classified into four groups: (i) PLWH with normal weight, (ii) PLWH 00420 from the Instituto de Salud Carlos III, Spain, co-financed by the European Regional with overweight/obesity, (iii) HIV-uninfected with normal weight, and (iv) HIV-uninfected with Development Fund. overweight/obesity. PLOS ONE | https://doi.org/10.1371/journal.pone.0252144 May 21, 2021 1 / 12 PLOS ONE FGF21, insulin resistance and obesity Competing interests: The authors have declared Insulin levels were higher in normal-weight PLWH than in the HIV-uninfected group but that no competing interests exist. not statistically significant, however, for the overweight/obesity PLWH group, insulin levels were significantly higher in comparison with the other three groups (p<0.0001). For FGF21, serum levels were slightly higher in the overweight/obesity groups in both patients and controls. In HIV-infected subjects, FGF21 levels showed a strong positive correlation with triglycer- ides, insulin levels and insulin resistance with a p-value <0.0001. In the seronegative group, FGF21 was only correlated with weight and waist circumference, showing an important association of FGF21 levels with the degree of obesity of the individuals. Conclusion Insulin resistance and FGF21 elevations were observed in overweight-obese PLWH. FGF21 elevation could be viewed as a compensation mechanism as, in the control group, FGF21 correlations appeared to be confined to weight and waist circumference. This can be explained based on the action of FGF21 promoting the uptake of glucose in adipose tissue. In PLWH, FGF21 was low, possibly as a result of a change in adiposity leading to a meta- bolic disruption. Introduction Combination antiretroviral therapy (cART) has significantly improved the prognosis of human immunodeficiency virus (HIV) infection, increasing the PLWH survival rate [1]. How- ever, prolonged exposure to cART may also cause adverse effects including mitochondrial tox- icity producing an increased risk of developing cardiovascular diseases, lipodystrophy, type 2 diabetes (T2D), insulin resistance (IR), lactic acidosis, among many others diseases [1–3]. Another metabolic alteration is obesity, which implies oxidative stress due to stored fat, and risk of insulin resistance due to the accumulation of reactive oxygen species (ROS) in adipose tissue and a deregulation in the production of adipokines [4–6]. At present, there are six main types of antiretroviral inhibitors: (i) nucleoside reverse tran- scriptase inhibitors (NRTIs), (ii) non-nucleoside reverse transcriptase inhibitor (NNRTIs), (iii) protease inhibitors (PIs), (iv) fusion inhibitors (FIs), (v) CCR5 co-receptor inhibitors, and (vi) integrase inhibitors (IIs), also known as integrase nuclear strand transfer inhibitors or (INSTIs) [7, 8]. Nucleoside and non-nucleoside inhibitors may prevent mitochondrial DNA (mtDNA) replication and repair by interfering with enzyme DNA-polymerase-y, which is sim- ilar to the reverse transcriptase of HIV. On the other hand, the NRTIs can strongly inhibit viral DNA elongation causing irreparable damage to mtDNA during replication, formation of ROS and a decrease in ATP production, causing cellular dysfunction [9]. PIs act directly on superoxide dismutase (SOD) and nicotinamide adenine dinucleotide phosphate (NADPH) enzymes, modifying their expression, generating ROS and causing intracellular and mitochon- drial damage [10]. It has been shown that PIs also promote abnormal glucose and lipid metab- olism with subsequent development of metabolic syndrome [11]. For example, lopinavir and ritonavir are drugs which have been reported to present an increased risk of metabolic toxicity [12]. Fibroblast growth factor 21 (FGF21) is a peptide hormone that is known to regulate glucose and lipid metabolism. FGF21 is expressed and secreted primarily in the liver and adipose PLOS ONE | https://doi.org/10.1371/journal.pone.0252144 May 21, 2021 2 / 12 PLOS ONE FGF21, insulin resistance and obesity tissue, promoting oxidation of glucose and fatty acids, and insulin sensitivity [13–15]. An increase in insulin levels has been observed in obesity and type 2 diabetes [16, 17]. As such, FGF21 therapy has been studied as a treatment against cardiovascular disease, in which improvements of adipogenesis, glucose regulation, and lipid metabolism have been previously reported [18]. People living with HIV are found to present a significant increase in FGF21 levels [19]. Sim- ilarly, elevated concentrations in FGF21 are found in patients suffering from a lipodystrophy condition, which may be associated with the development of IR, metabolic syndrome and car- diovascular disease [19]. Other studies performed in animals, particularly in rats, have also found elevated FGF21 protein levels and an overexpression in T2D, IR and lipodystrophy models [20]. The aim of the present study was to investigate the association between FGF21 protein levels with metabolic abnormalities and insulin resistance in Mexican Mestizo subjects living with HIV which were under cART treatment, placing special attention to the alterations in relation to the concurrence of overweight/obesity in people living with HIV. We hypothesized that FGF21 levels are higher in PLWH, and higher among PLWH with metabolic abnormalities. Materials and methods Patients In order to analyze the effect of obesity on FGF21 concentrations and metabolic disorders, 241 subjects, of which 160 PLWH were recruited from the Outpatient Centers for the Prevention and Care of AIDS and Sexually Transmitted Infections (CAPASITS) of Torreon, Coahuila and the Hospital Comprehensive Care Services (SAIH) of Gómez Palacio, Durango in Mexico (88 PLWH with normal weight and 72 PLWH with overweight/obesity). All subjects were treated with cART for at least one year. Exclusion criteria comprised patients with T2D, oncological dis- eases, or patients that had any kind of hormone therapy or antidiabetic agents. All participants were surveyed for sociodemographic and clinical data including age, place of residence, time of infection, and antiretroviral therapy data. Clinical records were consulted to obtain CD4+ cell counting and number of viral RNA copies. Inclusion criteria consisted of individuals 18–50 years old with a diagnosis of HIV infection, which were classified as the case group. Addition- ally, 81 unrelated HIV-uninfected subjects from the blood bank of these centers were included as a control group, (25 HIV-uninfected with normal weight and 56 HIV-uninfected with over- weight/obesity). Inclusion criteria comprised a serologic negative test for HIV, whereas exclu- sion criteria included individuals with metabolic disorders such as T2D, hypertension, dyslipidemia and oncological diseases. Both groups signed an informed consent letter where they agreed to participate in the study. This study was approved by the Bioethics Committee of the Faculty of Medicine at the Autonomous University of Coahuila in Torreon, Mexico. Anthropometric measurements Height, weight and waist circumference measurements of all participants were made and clas- sified according to their body mass index (BMI) in agreement with the World Health Organi- zation (WHO) criteria (kg/m2): (i) low weight (<18.5 kg/m2), (ii) normal weight (18.5–24.9 kg/m2), (iii) overweight (25–29.9 kg/m2) and (iv) obesity (� 30 kg/m2). Biochemical measurements Blood samples were collected under fasting conditions and centrifuged at 3000 RPM for 10 minutes to obtain serum, as well as measurements for glucose (mg/dL), total cholesterol (mg/ PLOS ONE | https://doi.org/10.1371/journal.pone.0252144 May 21, 2021 3 / 12 PLOS ONE FGF21, insulin resistance and obesity dL), high-density lipoprotein (HDL) (mg/dL), low-density lipoprotein (LDL) (mg/dL), very low-density lipoprotein (VLDL) (mg/dL), triglycerides (mg/dL) and atherogenic index. All measurements were analyzed by colorimetry in a Vitros1 250 automated dry chemistry ana- lyzer according to the manufacturer’s characteristics. Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) Insulin measurement was performed using the serum sample chemoluminescence method. The accepted reference values for the insulin hormone were 2–15 μUI/mL. Subsequently, the HOMA index calculation was performed to get the IR value [21]. Measurement of serum FGF21 The measurement of FGF21 levels was performed on the participants’ serum. Serum levels were determined in duplicate for each sample using an enzyme-linked immunoabsorbent assay (ELISA) specific for human FGF21 (Biovendor, Germany). The methodology was per- formed according to the manufacturer’s instructions. Statistical analysis Kolmogorov-Smirnov was performed in all continuous variables to test for normality. Socio- demographic and clinical data are presented as means, standard deviations and standard error for normally distributed variables whereas for non-normally distributed data are presented as medians. T-test was used to compare mean differences. For categorical data we used chi- squared test. For group comparisons, we used ANOVA with Tukey’s post hoc test. In addition, to examine the relationship among different variables we used Pearson correlation. Also, a multiple linear regression analysis was performed for significant associations after the univari- ate analysis. All results were considered significant with a value p<0.05. Finally, all calculations were performed using the SPSS statistical package version 19 (IBM Corp., Armonk, NY, USA) and the GraphPad Prism 6 package (GraphPad Software, Inc., San Diego, CA). Results The present study included 241 participants, 160 of whom were people with HIV/AIDS sero- positive and 81 HIV/AIDS uninfected individuals. Sociodemographic population data are listed in Table 1. Both groups were paired by sex (p = 0.16). As shown in Table 1, the majority (90.6%) of HIV/AIDS-seropositive subjects were given a nucleoside reverse transcriptase inhibitor, followed by non-nucleoside reverse transcriptase and protease inhibitors with 56.8% and 40.6% respectively. In addition, the average viral load and average CD4+ cell for this group are also shown in Table 1. The patients with undetectable viral load represented 80.3% of studied HIV-seropositive population. According to metabolic parameters, PLWH showed higher levels of triglycerides, VLDL cholesterol, insulin and HOMA-IR than controls, and lower for total cholesterol, LDL cholesterol and FGF21. For body composition, BMI was con- siderably higher in controls than in PLWH (Table 1). To analyze the biochemical parameters and their relation with the obesity, the participants were classified into four groups: (i) PLWH with normal weight, (ii) PLWH with overweight/ obesity, (iii) HIV-uninfected with normal weight, and (iv) HIV-uninfected with overweight/ obesity, based on BMI classification. All seropositive individuals included in the study had a regime of cART of at least one year, with a mean of 6.42 years under treatment (Table 1). As shown in Table 2, most measurements demonstrated significant differences among the four groups, particularly those related to weight, BMI, waist perimeter, VLDL, LDL and PLOS ONE | https://doi.org/10.1371/journal.pone.0252144 May 21, 2021 4 / 12 PLOS ONE FGF21, insulin resistance and obesity Table 1. Demographic and clinical characteristics of the study population. PLWH Controls p-value n = 160 n = 81 Sex n (%) n (%) Male 124 (77.5) 56 (69.1) 0.16a Female 36 (22.5) 25 (30.8) Age 39.78 ±10.78 32.45 ± 10.61 <0.0001b Duration with HIV infection (years) 7.05 ±6.37 cART NRTI 145 (90.6%) NNRTI 91 (56.8%) II 4 (2.5%) PI 65 (40.6%) M±SD cART (years) 6.42±5.83 Median Min Max HIV-RNA (copies mL) 39 20 416,107 CD4+ cell counting (cell uL) 452 3 1280 Body composition Mean (SE) Weight 72.13 (1.1) 81.09 (2.2) <0.0001b Height 1.66 (0.01) 1.69 (0.01) 0.112b BMI 25.40 (0.4) 28.14 (0.7) 0.0006b Waist perimeter (cm) 90.84 (1.1) 92.66 (1.8) 0.367b Metabolic parameters Glucose (mg/dL) 89.78 (2.0) 92.10 (1.5) 0.455b Total cholesterol (mg/dL) 174.2 (3.0) 190.3 (6.2) 0.022b Triglycerides (mg/dL) 218.5 (16.5) 148.7 (18.3) 0.009b HDL cholesterol (mg/dL) 45.96 (1.0) 48.96 (1.4) 0.098b VLDL cholesterol (mg/dL) 36.85 (1.6) 26.17 (1.4) <0.0001b LDL cholesterol (mg/dL) 97.44 (2.4) 115.6 (5.5) <0.0001b IA 4.03 (0.1) 3.99 (0.1) 0.778b Insulin (Ul) 10.70 (0.5) 5.63 (0.5) <0.0001b HOMA-IR 2.53 (0.1) 1.35 (0.1) <0.0001b FGF21 (pg/mL, log) 2.31 (0.04) 2.56 (0.02) <0.0001b State of residence n (%) n (%) Coahuila 75 (46.9) 50 (61.7) Durango 66 (41.2) 31 (38.3) Others 19 (11.9) NRTI: nucleoside reverse transcriptase inhibitor, NNRTI: non-nucleoside reverse transcriptase inhibitor, II: integrase inhibitor and PI: protease inhibitor. a Chi-squared test and b t-student test. SD: standard deviation, SE: standard error. https://doi.org/10.1371/journal.pone.0252144.t001 HOMA-IR which showed the highest differences with a p-value<0.0001. Interestingly, insulin levels were higher in normal-weight PLWH cases than in the overweight/obesity uninfected group (7.9 vs 6.5), although not statistically significant. However, for the overweight/obesity PLWH group the differences in insulin levels were significant in comparison with the other three groups (p<0.0001). For FGF21, protein levels were slightly higher in the overweight/obe- sity groups in both patients and controls. PLOS ONE | https://doi.org/10.1371/journal.pone.0252144 May 21, 2021 5 / 12 PLOS ONE FGF21, insulin resistance and obesity Table 2. Clinical and biochemical parameters in PLWH and HIV-uninfected individuals grouped by BMI classification. Controls PLWH Normal weight Overweight/Obesity Normal weight Overweight/Obesity p-value n = 25 n = 56 n = 88 n = 72 Weight 68.0 87.1 aaaa 62.7 bbbb 83.5 aaaa cccc <0.0001 Height 1.74 1.67 1.66 1.67 0.093 Body composition (BMI) 22.3 30.8 aaaa 21.7 bbbb 29.8 aaaa cccc <0.0001 Waist perimeter (cm) 80.2 98.7 aaaa 83.2 bbbb 100.4 aaaa cccc <0.0001 Glucose (mg/dL) 87.1 94.4 87.1 93.0 0.393 Total cholesterol (mg/dL) 193.4 188.4 169.9 179.5 0.035 Triglycerides (mg/dL) 103 170.2 191.1 251.9 aa 0.005 HDL cholesterol (mg/dL) 53.2 46.9 47.8 43.6 0.010 VLDL cholesterol (mg/dL) 20.6 28.8 32.8 41.6 bb aaa <0.0001 LDL cholesterol (mg/dL) 119.5 113.3 89.1 b 94.1 0.0001 IA 3.7 4.09 3.80 b 4.3 0.046 Insulin (Ul) 3.6 6.5 7.9 14.1 aaaa bbbb cccc 0.0001 HOMA-IR 0.80 1.62 1.84 3.37 aaaa bbbb cccc 0.0005 FGF21 (pg/mL, log) 2.53 2.57 2.25 bbbb 2.38 b 0.0003 Individuals were classified according to their body mass index (BMI): normal weight <25 BMI and overweight/ obesity �25 BMI; HDL: high-density lipoprotein; LD: low-density lipoprotein; VLDL: very low-density lipoprotein; IA: atherogenic index; IR: insulin resistance; FGF21, fibroblast growth factor 21. Differences between groups were analyzed by ANOVA followed by Tukey’s post hoc test. Significance: a: p<0.05 vs normal-weight control, b: p<0.05 vs obesity control, c: p<0.05 vs normal-weight HIV+; aa: p<0.01, aaa: p<0.001, aaaa: p<0.0001. FGF21 values were logarithmically transformed for statistical analysis. https://doi.org/10.1371/journal.pone.0252144.t002 In the univariate analysis, the highest correlations for PLWH subjects were observed for tri- glycerides, insulin levels and insulin resistance with a p-value <0.0001 for these three variables (Table 3 and Fig 1). In contrast, in the HIV-uninfected group, no correlations were found in lipid profile or in hydrocarbon metabolisms such as glucose and insulin. The only correlation was found in relation to the weight, and waist circumference, showing an important associa- tion of FGF21 protein with the degree of obesity of the individuals (Table 4). Table 3. Univariate analysis for FGF21 and biochemical parameters in PLWH. Correlation R p-value FGF21-Glucose 0.23 0.003 FGF21-Cholesterol 0.09 0.253 FGF21-Triglycerides 0.32 <0.0001 FGF21-HDL -0.15 0.06 FGF21-VLDL 0.27 0.0005 FGF21-LDL -0.008 0.916 FGF21-IA 0.23 0.002 FGF21-Insulin 0.32 <0.0001 FGF21-IR 0.36 <0.0001 FGF21-Weight 0.13 0.090 FGF21-BMI 0.07 0.330 FGF21-WP 0.14 0.087 FGF21: fibroblast growth factor 21; HDL: high-density lipoprotein; LDL: low-density lipoprotein; VLDL: very low- density lipoprotein; IA: atherogenic index; IR: insulin resistance; BMI: body mass index; WP: waist perimeter. Significant values after Pearson’s correlation are shown in bold. https://doi.org/10.1371/journal.pone.0252144.t003 PLOS ONE | https://doi.org/10.1371/journal.pone.0252144 May 21, 2021 6 / 12 PLOS ONE FGF21, insulin resistance and obesity Fig 1. Significant correlations between insulin resistance levels and fibroblast growth factor-21 (FGF21) levels in PLWH. https://doi.org/10.1371/journal.pone.0252144.g001 Finally, after a multivariable analysis, insulin and triglycerides showed a statistically signifi- cant correlation with serum FGF21 levels in HIV-seropositve individuals (p = 0.002 and p = 0.005, respectively) (Table 5). Discussion The present study shows the already reported trend of worsened metabolic profile (increased glycemia, hyperinsulinemia, hypertriglyceridemia, HOMA-IR index) in PLWH, comparing these parameters with HIV-uninfected individuals stratified by BMI. Also, consistently with Table 4. Univariate analysis for FGF21 and biochemical parameters in HIV-uninfected individuals. Correlation R p-value FGF21-Glucose 0.11 0.326 FGF21-Cholesterol 0.08 0.460 FGF21-Triglycerides -0.09 0.410 FGF21-HDL -0.20 0.066 FGF21-VLDL 0.07 0.506 FGF21-LDL -0.06 0.550 FGF21-IA 0.07 0.530 FGF21-Insulin 0.18 0.102 FGF21-IR 0.17 0.118 FGF21-Weight 0.26 0.016 FGF21-BMI 0.17 0.125 FGF21-WP 0.33 0.003 FGF21: fibroblast growth factor 21; HDL: high-density lipoprotein; LDL: low-density lipoprotein; VLDL: very low- density lipoprotein; IA: atherogenic index; IR: insulin resistance; BMI: body mass index; WP: waist perimeter. Significant values after Pearson’s correlation are shown in bold. https://doi.org/10.1371/journal.pone.0252144.t004 PLOS ONE | https://doi.org/10.1371/journal.pone.0252144 May 21, 2021 7 / 12 PLOS ONE FGF21, insulin resistance and obesity Table 5. Multivariable analysis for FGF21 and biochemical parameters in HIV- seropositive individuals. Model Beta CI 95% p-value FGF21-Glucose 0.094 (-0.695; 2.810) 0.235 FGF21-Triglycerides 0.225 (0.095; 0.538) 0.005 FGF21-Insulin 0.244 (3.703; 15.605) 0.002 FGF21: fibroblast growth factor 21; CI: confidence interval. Significant values after multiple linear regression are shown in bold. https://doi.org/10.1371/journal.pone.0252144.t005 previous data, HIV-infected patients showed also a similar trend of metabolic abnormalities even if showing normal weight. The occurrence of overweight/obesity in PLWH worsened dramatically the metabolic profile. This is especially dramatic for the HOMA-IR index, which were highly increased in patients combining the HIV-infection and overweight/obese condi- tions. This indicated the high trend to insulin resistance and subsequent metabolic derange- ments when HIV-infected patients bear an obese condition. Fibroblast growth factor 21, FGF21, is an endocrine factor that regulates metabolic homeo- stasis, and is mainly expressed in the liver. FGF21 has the potential to activate glucose uptake in adipocytes [22], being also studied in mice models to show its role in the inflammatory state of obesity [23–26]. In humans, high levels of serum FGF21 have been found in people with obesity, particularly when metabolic complications are present such as type 2 diabetes [17, 27, 28]. FGF21 high levels have also been associated with cardiovascular risk [17]. In PLWH with symptoms of lipodystrophy, serum FGF21 appears increased [13, 19, 29]. Thus, the purpose of this study was to analyze FGF21 protein levels in PLWH and to examine its relationship with metabolic changes such as insulin resistance, lipid profile disorders and inflammatory factors, paying special attention to the obesity variable. Biochemical measurements showed significant differences between PLWH and unin- fected-HIV in the lipid profile, particularly in HIV-positive groups (normal and over- weight/obesity), which were similar to those found in previous studies [19, 30]. cART has been associated with dyslipidemia, which in part explains the high risk of cardiovascular complications in PLWH. Although, there are different treatments using statins for meta- bolic disorders such as hypercholesterolemia, these may have interactions with antiretrovi- ral therapy in the HIV management. For example, it has been shown that some PIs and NNRTIs along with statins are metabolized by cytochrome P450 which may result in risk of intolerance [30, 31]. Insulin resistance levels in the PLWH were considerably higher compared to other HIV seronegative groups. Other studies have also reported insulin resistance in HIV-positive sub- jects [19, 30]. However, these previous studies did not mention the frequency or prevalence of insulin resistance. Here, we observed that 39% of the PLWH had IR. The analysis of FGF21 showed that this protein had an increase in the control group vs. PLWH. The stratification by BMI may explain why in our results, overweight/obesity groups had higher levels of FGF21 protein, particularly in the control group; however; the number of controls used in the study may be a limitation. Therefore, it is suggested to increase the num- ber of controls to validate these results. According to other studies, FGF21 levels were increased in a state of obesity, regardless of the degree of insulin resistance [32], although these previous studies included a patient population with a highest rate of obesity (BMI mean ~40) whereas in the current study population of overweight/obese patients averaged a BMI mean around 30. Mouse model studies and even some human studies indicated that pharmacologi- cal treatment with FGF21 can attenuate comorbidities associated with obesity [33]. Therefore, PLOS ONE | https://doi.org/10.1371/journal.pone.0252144 May 21, 2021 8 / 12 PLOS ONE FGF21, insulin resistance and obesity FGF21 elevation in an overweight-obese state could be viewed as a compensation mechanism or even the possibility of having resistance to FGF21.Studies performed in animal models that attempted to corroborate the signaling pathways of FGF21 and its association with insulin resistance showed that plasma elevation was due to overregulation in the liver. However, the lipodystrophic models did not allow compensation for glucose uptake and insulin resistance [20]. The present study confirmed that there is a compensation factor that may be brought by the FGF21 protein because, in the overweight/obesity control group, the insulin level remains at 6.5 UI compared to 14.1 UI observed in the overweight/obesity experimental group. FGF21 appears to act in a paracrine model to increase glucose uptake in low insulin conditions. A pre- vious study in patients with acantosis nigricans showed an increased level of FGF21, however, the insulin levels were normal, indicating a possible compensatory response [34]. A similar trend was also seen in the present study in the control group. It is suggested that metabolic changes may be in relation to lipid metabolism and choles- terol levels, triglycerides, especially in subjects with HIV [13]. This association was also observed in this study showing important correlations with lipid profile levels as shown in Table 3. On the other hand, there is evidence which suggests that cardiovascular disease is associated with increased levels of FGF21 [18, 35], therefore, the results found in this study showed a relationship between FGF21 and dyslipidemia in the HIV-positive population. In the control group, FGF21 correlations appeared confined to the weight and waist cir- cumference of the participants. This can be explained based on the action of this factor on the uptake of glucose in adipose tissue in the case of obesity or overweight [17], the elevation of FGF21 can be seen as a consequence. However, in the HIV-seropositive group, FGF21 was low, possibly as a result of a change in adiposity leading to a metabolic disruption. In addition, antiretroviral therapy has been associated for a long time with disorders such as lipodystrophy which causes a disorder in the adipose tissue distribution, mitochondrial dysfunction and alterations in adipocyte lipolysis [13]. There are multiple signaling pathways involving FGF21 such as associations with the peroxisome proliferator-activated receptors (PPARs). PPAR-γ is a transcriptional regulator of adipogenesis and inducible by FGF21 [36]. PPAR-α affects hepatic regeneration [37] and, in conjunction with the CREBH element, binds protein function as binary transcrip- tional activators to regulate lipid metabolism thus activating FGF21 [16]. Therefore, it is important to evaluate in the future the interaction of these signaling pathways and their relationship with metabolic changes in HIV-positive patients. In summary, circulating lev- els of FGF21 are associated with metabolic disturbances in PLWH, including insulin resis- tance, whereas in uninfected patients, FGF21 levels were mainly related to markers of visceral adiposity. Supporting information S1 Data. (PDF) Acknowledgments We thank the subjects for their participation in this study Dr. Maria de Jesus Vaquera Rivera as Director of the Outpatient Center for aids prevention and care and sexually transmitted infections (CAPASITS) and Dr. Enrique Castañeda Guerrero from the General Hospital of the Comprehensive Care Service of the Secretary of Health for the care of people living with HIV (SAI) for providing HIV-related data. PLOS ONE | https://doi.org/10.1371/journal.pone.0252144 May 21, 2021 9 / 12 PLOS ONE FGF21, insulin resistance and obesity Author Contributions Conceptualization: Arguiñe Ivonne Urraza-Robledo, Marta Giralt, Francesc Villarroya, Fran- cisco Carlos López-Márquez. Data curation: Arguiñe Ivonne Urraza-Robledo, Faviel Francisco González-Galarza, Pablo Ruiz Flores. Formal analysis: Arguiñe Ivonne Urraza-Robledo, Faviel Francisco González-Galarza, Peré Domingo. Investigation: Arguiñe Ivonne Urraza-Robledo, Marta Giralt, Francesc Villarroya. 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