PCB-126 exposure promotes brown adipose tissue dysfunction and metabolic inflexibility in mice

Abstract

Polychlorinated biphenyls (PCBs) remain a global health concern due to their persistence and toxicity. PCB-126, a potent aryl hydrocarbon receptor (AhR) agonist, is linked to metabolic disruption, yet its impact on brown adipose tissue (BAT) is not fully understood. Male C57BL/6 mice were exposed to PCB-126 (5 µmol/kg) for 10 weeks, followed by morphological, biochemical, and molecular interscapular brown adipose tissue (iBAT) analyses. Despite comparable energy intake and delta body weight, PCB-126 markedly increased relative visceral fat and reduced relative iBAT mass. Oral glucose tolerance testing revealed impaired glucose handling, with higher glycemia across the curve and a ~ 30% increase in AUC, indicating systemic metabolic dysfunction. Histology showed extensive lipid droplet remodeling—reduced lipid area fraction but increased droplet number—consistent with a blunted thermogenic phenotype. At the molecular level, PCB-126 downregulated thermogenic markers (Ucp1, Prdm16, Pgc-1α, Adrb3) and Vegfa, reflecting impaired vascularization. Genes involved in lipid droplet regulation (Cidea), adipogenic control (Pparγ), and energy-sensing (Ampk2) were also suppressed, reinforcing thermogenic loss and reduced metabolic flexibility. These changes coincided with heightened inflammation (Tnf-α, Il-6), oxidative stress (↑MDA, ↑NOx), and compromised antioxidant defenses (↓SOD, ↓catalase, ↑GPx). Overall, PCB-126 disrupts BAT structure and transcriptional programming, impairs glucose tolerance, and promotes visceral fat accumulation through combined thermogenic, inflammatory, and redox dysregulation.

Introduction

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that, despite being banned for decades, continue to represent a global health concern^1,2. Their environmental stability and lipophilic nature allow them to accumulate in ecosystems and in the food chain, leading to prolonged exposure in humans and animals³. Among the various congeners, PCB-126 is one of the most toxic, acting through activation of the aryl hydrocarbon receptor (AhR) and triggering a cascade of cellular and metabolic disturbances^4,5. Epidemiological and experimental evidence has linked PCB exposure to endocrine disruption, alterations in glucose and lipid metabolism, and increased risk of chronic diseases, reinforcing the urgency of investigating their biological effects⁶. In this context, understanding how PCB-126 interferes with key metabolic tissues becomes essential to clarify its contribution to the burden of modern metabolic disorders.

Brown adipose tissue (BAT) has gained prominence in recent years as a metabolically active organ capable of dissipating energy in the form of heat through UCP1-mediated thermogenesis^7,8. Beyond its role in adaptive thermogenesis, BAT exerts systemic benefits by improving glucose homeostasis and lipid clearance, thereby contributing to metabolic health. Unlike white adipose tissue, which primarily stores energy, BAT actively consumes substrates, positioning it as a protective tissue against obesity and its complications^5,9,10. However, this high metabolic activity also makes BAT particularly vulnerable to environmental insults and xenobiotics that may compromise its function and reduce its contribution to energy balance⁷.

When BAT becomes dysfunctional, its thermogenic capacity declines, leading to impaired energy expenditure, ectopic lipid deposition, and systemic metabolic stress. Disruption of BAT function has been associated with insulin resistance, dyslipidemia, and the progression of metabolic syndrome¹³. Experimental findings suggest that environmental pollutants, including dioxin-like PCBs¹¹, may interfere with adipocyte differentiation, mitochondrial activity, and redox balance, processes that are central to BAT physiology¹². Yet, the specific impact of PCB-126 on BAT morphology and functionality remains poorly characterized, representing a critical gap in the current understanding of pollutant-induced metabolic dysfunction.

Building upon this background, the present study was designed to evaluate the effects of PCB-126 exposure on BAT structure and thermogenic function. We hypothesized that PCB-126 induces morphological and molecular changes consistent with impaired lipid handling and reduced thermogenic potential. By integrating histological, biochemical, and molecular approaches, our aim was to shed light on how this pollutant interferes with a tissue that plays a pivotal role in systemic energy metabolism, ultimately providing new insights into the mechanisms by which environmental contaminants contribute to metabolic disease.

Results

The experimental design and an overview of the primary metabolic parameters evaluated following chronic PCB-126 exposure are presented in Fig. 1. The average food intake and body weight change over the 10-week experimental period were similar across groups (Fig. 2A and B). The glucose curve over time revealed that mice exposed to PCB-126 displayed consistently higher plasma glucose concentrations at all measured time points (0, 30, 60, 90, and 120 min) compared with the control group (Fig. 2C). Analysis of the area under the curve (AUC) further supported this finding: the PCB-126 group exhibited a significantly greater AUC relative to controls (p < 0.05; Fig. 2D). This increase in AUC demonstrates impaired glucose clearance in PCB-126–exposed mice, highlighting a defect in whole-body glucose homeostasis.

Fig. 1.

Experimental design. Three-month-old male mice were divided into a group that received PCB 126 (n = 10) (5 µmol/kg body weight, dissolved in corn oil) and a group that received a vehicle (n = 10) (corn oil alone). Treatments were administered by intragastric gavage at weeks 2, 4, 6, and 8 of the experimental protocol. After 10 weeks of experimentation, the animals were euthanized, the plasma was separated for biochemical analyses, and iBAT was used for histological, protein, and gene expression analyses.

Fig. 2.

Nutritional and metabolic profile of the experimental groups. Mean food intake (A); body weight progression up to 10 weeks (B); fasting glucose levels (C); area under the glucose tolerance curve (D); visceral fat mass (E); and interscapular brown adipose tissue (iBAT) mass (F). Data are expressed as mean ± SD (n = 10 per group). Area under the curve (AUC) for the glucose tolerance test was calculated using the trapezoidal method from blood glucose values measured at 0, 15, 30, 60, and 120 min after glucose administration. Statistical differences between groups were determined using the Student’s t-test (p < 0.05).

However, mean visceral fat mass (VFM) was significantly higher in the PCB-126 group compared with the Control (p < 0.05; Fig. 2E). In contrast, iBAT mass normalized to body weight was reduced in the PCB-126 group relative to the Control (p < 0.05; Fig. 2F).

Additionally, mice exposed to PCB-126 showed a significant rise in blood glucose levels (p < 0.05; Table 1). Plasma insulin concentrations were also higher (p < 0.05), leading to an increase in HOMA-IR (p < 0.05). These findings indicate that PCB-126 exposure impairs glucose homeostasis, promoting insulin resistance. Plasma ALT activity in the PCB-126 group was almost six-fold higher than in controls (p < 0.05), while AST increased by + 172% (p < 0.05; Table 1). This pronounced elevation in transaminases points to marked hepatocellular damage associated with PCB-126 exposure. Total cholesterol levels did not differ significantly between groups, although a 16% reduction was noted in the exposed group. By contrast, plasma triglycerides were significantly elevated in the PCB-126 group (p < 0.05), highlighting lipid metabolic dysregulation. PCB-126 exposure triggered a robust pro-inflammatory response, evidenced by higher circulating IL-6 (p < 0.05) and TNF-α (p < 0.05; Table 1). These changes reflect an exacerbated low-grade inflammatory state.

Table 1.

Evaluation of glucose–insulin homeostasis, hepatic enzymes, lipid profile, and inflammatory cytokines in plasma of mice chronically exposed to PCB-126.

Biomarkers	CON	PCB-126
Glucose (mg/dl)	118.6b ± 18.7	188.9a ± 25.3
Insulin (µIU/ml)	13.0 b ± 1.5	18.0 a ± 4.3
HOMA-IR	6.1 b ± 2.0	8.4 a ± 1.7
ALT (U/L)	9.6 b ± 4.1	56.8 a ± 16.1
AST (U/L)	70.4 b ± 26.9	191.8 a ± 8.1
Cholesterol (mg/dL)	56.7 ± 7.9	47.7 ± 7.6
Triglyceride (mg/dL)	32.5 b ± 5.5	54.6 a ± 10.9
IL-6 (pg/ml)	45.4 b ± 3.3	73.7 a ± 3.8
TNF-α (pg/ml)	14.0 b ± 1.5	20.0 a ± 1.0

CON = control group; PCB 126 = polychlorinated biphenyl-126, ALT = alanine aminotransferase, AST = aspartate aminotransferase, TAG = triglycerides, IL-6 = interleukin 6, TNF-α = tumor necrosis factor-alpha. Statistical differences between groups (different letters) were determined using the Student’s t-test (p < 0.05).

To further investigate the effects of PCB-126 on iBAT metabolism, we measured interscapular body temperature (Fig. 3A). Although PCB-126–exposed animals showed a modest but significant increase in core temperature (p < 0.05; Fig. 3B), iBAT displayed molecular and histological signatures consistent with reduced thermogenic competency. The Control group exhibited preserved architecture, characterized by multilocular adipocytes containing small to medium lipid droplets distributed homogeneously throughout the cytoplasm (Fig. 3C). In sharp contrast, PCB-126 exposure induced evident alterations, with a greater number of smaller lipid droplets, conferring a fragmented and heterogeneous cytoplasmic appearance. Quantitative analyses confirmed these observations: the percentage of cytoplasmic area occupied by lipid droplets was significantly reduced in the PCB-126 group (p < 0.05; Fig. 3D), while the number of droplets per field was markedly higher (p < 0.05; Fig. 3E). These findings indicate a remodeling of lipid storage, whereby total lipid load remained comparable between groups, but PCB-126 triggered droplet fragmentation and increased droplet number. Such structural alterations were accompanied by impaired thermogenic capacity. Immunofluorescence demonstrated a clear reduction in UCP1 staining in PCB-126-exposed animals compared to Control (Fig. 3F), a finding further supported by gene expression analysis showing a significant downregulation of Ucp1 (p < 0.05; Fig. 3G). Similarly, Adrb3, a marker of adrenergic responsiveness and thermogenic activation, was also reduced in the PCB-126 group (p < 0.05; Fig. 3H). Together, these results indicate that PCB-126 exposure compromises not only the morphology but also the functional integrity of brown adipose tissue, blunting its metabolic responsiveness.

Fig. 3.

Chronic PCB-126 exposure disrupts interscapular brown adipose tissue structure, thermogenic markers, and redox-sensitive pathways. Infrared thermographic images of the interscapular region in Control and PCB-126–exposed mice showing surface temperature distribution as a proxy of brown adipose tissue (BAT) activity (A), with corresponding quantitative analysis of interscapular temperature (B). Representative hematoxylin and eosin (H&E)–stained sections of interscapular BAT (iBAT) illustrating tissue morphology (C). Stereological quantification of lipid droplet area fraction (D) and lipid droplet number per tissue area (E). Representative immunofluorescence images of uncoupling protein 1 (UCP1; green) with nuclear counterstaining (DAPI; blue) in iBAT (F). Relative mRNA expression of Ucp1 (G) and Adrb3 (H) in iBAT. Gene expression data were normalized to Gapdh. Data are presented as mean ± SD (n = 5 animals per group). Statistical comparisons between groups were performed using Student’s t-test. Different letters indicate statistically significant differences (p < 0.05).

At the transcriptional level, PCB-126 consistently disrupted the gene regulatory network governing thermogenesis. Expression of Prdm16 (Fig. 4A), the master regulator of brown adipocyte identity and biogenesis, was significantly reduced (p < 0.05), paralleled by decreased expression of Cidea (p < 0.05; Fig. 4B) and Ppargc1a (p < 0.05; Fig. 4C), key drivers of mitochondrial biogenesis and thermogenic activation. Expression of Pparα (Fig. 4D) and Ampk2 (Fig. 4E), both essential for lipid metabolism and energy sensing, was also diminished in the PCB-126 group (p < 0.05). In contrast, Cytc (Fig. 4F) expression remained unaltered, suggesting that the pollutant’s effects were not uniformly distributed across the mitochondrial machinery. Moreover, Vegfa expression (Fig. 4G) was reduced (p < 0.05) in PCB-126-treated animals, indicating impaired vascularization and oxygen supply, processes critical for sustaining thermogenesis.

Fig. 4.

Transcriptional profile of thermogenic, mitochondrial, and inflammatory genes in interscapular brown adipose tissue (iBAT). iBAT gene expression of Prdm16 (A), Cidea (B), Pgc1α (C), Pparα (D), Ampk2 (E), Ppargc1a (F), Vegfa (G), Tnfα (H), and Il6 (I). Data are expressed as mean ± SD (n = 5 per group). Statistical differences between groups were determined using the Student’s t-test (p < 0.05).

Within the inflammatory axis, significant increases (p < 0.05) in Tnfα (Fig. 4H) and Il-6 (Fig. 4I) expression were observed in PCB-126-exposed mice compared with Controls, suggesting that exposure not only suppressed the thermogenic cascade but also promoted a pro-inflammatory milieu that may exacerbate metabolic dysfunction in brown fat.

Finally, PCB-126 exposure induced a clear redox imbalance in iBAT. Treated animals exhibited significantly elevated levels of MDA (p < 0.05; Fig. 5A), a marker of lipid peroxidation, and increased nitrite/nitrate (NOx) concentrations (p < 0.05; Fig. 5B), reflecting enhanced oxidative and nitrosative stress relative to Controls. In parallel, endogenous antioxidant defenses were consistently impaired: activities of SOD (p < 0.05; Fig. 5C) and catalase (p < 0.05; Fig. 5D) were markedly reduced, while GPx activity (Fig. 5E) was increased (p < 0.05), possibly reflecting a compensatory but insufficient response. Collectively, this pattern demonstrates that PCB-126 not only amplifies oxidative processes but also weakens the antioxidant defense system, favoring a pro-oxidative environment within brown adipose tissue.

Fig. 5.

Oxidative stress and antioxidant defense markers in interscapular brown adipose tissue (iBAT). Malondialdehyde (MDA) (A); nitrite (NO₂) (B); superoxide dismutase (SOD) (C); catalase (CAT) (D); and glutathione peroxidase (GPx) (E). Data are expressed as mean ± SD (n = 5 per group). Statistical differences between groups were determined using the Student’s t-test (p < 0.05).

Discussion

Despite comparable energy intake and body weight trajectories across groups over the 10-week period, the increase in visceral fat mass (VFM) in the PCB-126 group, alongside the reduction in relative iBAT mass, suggests a redistribution of energy balance toward visceral storage when adaptive thermogenesis is impaired. A plausible mechanism involves activation of the aryl hydrocarbon receptor (AhR) by PCB-126, a dioxin-like congener, which represses the thermogenic program. Indeed, dioxin-like pollutants are well-documented to inhibit browning and reduce UCP1 expression^13,14, while AhR deficiency protects against adiposity and promotes multilocular/thermogenic phenotypes¹⁵, reinforcing a causal link between PCB-126 and AhR signaling and the inactivation of thermogenic adipose tissue⁵.

Although iBAT dysfunction likely contributes to impaired glucose clearance following PCB-126 exposure, the systemic metabolic phenotype cannot be attributed to iBAT alterations alone. The marked elevation of transaminases, together with hypertriglyceridemia, hyperinsulinemia, and increased HOMA-IR, strongly indicates hepatic involvement, consistent with known PCB-126 accumulation in the liver and AhR-mediated disruption of hepatic glucose and lipid metabolism^16,17. Reduced BAT-mediated substrate uptake may further increase glucose and lipid flux to the liver, exacerbating hepatic metabolic stress, while hepatic dysfunction and systemic inflammation may, in turn, impair iBAT thermogenic responsiveness. In parallel, selective expansion of visceral white adipose tissue suggests altered energy partitioning and potential WAT dysfunction, and skeletal muscle may also be indirectly affected by increased substrate availability and mitochondrial stress¹⁸. Collectively, these findings support a multi-organ model in which PCB-126 disrupts coordinated metabolic regulation across iBAT, liver, and white adipose tissue, leading to whole-body metabolic inflexibility.

Collectively, these findings indicate that chronic PCB-126 exposure is associated with marked structural and molecular remodeling of iBAT, accompanied by alterations in systemic metabolic parameters. While iBAT is a well-established regulator of whole-body energy homeostasis, the present data should be interpreted as supporting an association between BAT dysfunction and systemic metabolic disturbances, rather than establishing direct causality. Thus, iBAT impairment likely represents an important contributing component within a broader network of PCB-126–induced metabolic effects.

At the morphological level, iBAT from PCB-126–exposed mice displayed lipid droplet remodeling, characterized by a lower fraction of area occupied by lipids but a higher number of droplets, consistent with depot-specific reorganization and thermogenic deactivation. In models where the thermogenic program is suppressed (through AhR activation or loss of adrenergic drive), such droplet al.terations typically parallel reductions in UCP1 and key brown-fat regulators⁵. In this context, the PRDM16–PGC-1α axis emerges as a critical determinant of brown adipocyte identity^5,19. PRDM16 orchestrates the induction of PGC-1α/PGC-1β and thermogenic genes (including Ucp1 and Cidea)⁷. Thus, the observed downregulation of Prdm16, Ppargc1a (PGC-1α), and Cidea provides a direct mechanistic framework for the attenuated iBAT phenotype.

Functionally, reductions in UCP1 (both protein and mRNA) and Adrb3 expression point to impaired β-adrenergic responsiveness, the central driver of thermogenesis via the cAMP/PKA-p38-PGC-1α-UCP1 pathway. In both rodents and humans, β3-AR is required to maintain lipolytic and thermogenic machinery; its downregulation is associated with inflammatory catecholamine resistance^5,20. TNF-α is a well-established contributor to this desensitization, consistent with the present findings of increased Tnfα/Il-6 expression alongside reduced Adrb3^5,13,21.

The AMPK pathway converges on this phenotype. AMPK preserves mitochondrial homeostasis in iBAT and promotes browning/thermogenesis; thus, reduced Prkaa2 (AMPK2) expression aligns with the declines in PGC-1α and UCP1, indicating compromised thermogenic capacity. The demonstrated coupling between β-adrenergic signaling and AMPK activation in iBAT further supports a synergistic effect of Adrb3 and AMPK downregulation in suppressing thermogenesis^22–26. Lipid metabolism was also disrupted, with reduced Pparα expression being particularly significant. PPARα induces PGC-1α and PRDM16 and sustains fatty acid oxidation and UCP1 expression; its downregulation therefore closes the loop of negative regulation on the oxidative-thermogenic program.

At the angiogenic level, the marked reduction of Vegfa in iBAT following PCB-126 exposure is highly important. VEGF-A is essential for vascularization, oxygen delivery, and thermogenic capacity of adipose tissue^18,27,28. Loss of VEGF-A has been directly linked to capillary rarefaction and iBAT whitening²⁹. Thus, reduced Vegfa expression likely contributes to functional and mitochondrial hypoxia as well as sustained suppression of Ucp1/PGC-1α³⁰.

The inflammatory and redox milieu provides additional reinforcement. Elevated MDA and NOx levels, together with reduced antioxidant defenses (SOD, GPx, catalase), indicate exacerbated oxidative stress and impaired antioxidant capacity. PCB-126 is recognized as a pro-oxidant in various tissues and models³¹, and reduced UCP1 itself may amplify ROS generation by limiting protective mitochondrial uncoupling. This self-perpetuating cycle, in which AhR activation leads to the downregulation of UCP1, PGC-1α, PPARα, AMPK, and VEGFA, results in mitochondrial dysfunction and hypoxia³². These alterations promote the accumulation of reactive oxygen species, which in turn drive inflammation mediated by TNF-α and IL-6³³. The inflammatory environment contributes to β-adrenergic resistance through the reduction of Adrb3 expression³⁴. Altogether, this sequence of events coherently explains the hypoactive phenotype of brown adipose tissue and the concomitant expansion of visceral fat observed. Finally, converging evidence from both in vitro and in vivo studies shows that PCB-126 inhibits adipogenesis, suppresses browning, and induces mitochondrial dysfunction, supporting the broader generalizability of the mechanisms identified here across diverse cellular contexts (Fig. 6).

Fig. 6.

Proposed mechanism of PCB-126–induced brown adipose tissue dysfunction and whitening. Chronic exposure to PCB-126 activates the aryl hydrocarbon receptor (AhR), triggering a transcriptional reprogramming in brown adipose tissue (BAT). This activation leads to the suppression of key thermogenic regulators, including PRDM16, PGC-1α, and UCP1, resulting in reduced mitochondrial oxidative capacity. In parallel, VEGFA-mediated vascularization is impaired, compromising oxygen and nutrient delivery to brown adipocytes. Concomitantly, PCB-126 exposure enhances inflammatory and oxidative stress pathways, characterized by elevated expression of TNF-α and IL-6, further disrupting BAT homeostasis. Together, these molecular and cellular alterations promote BAT whitening, reflected by increased lipid droplet accumulation and decreased thermogenic activity. The combined loss of thermogenic, vascular, and redox balance culminates in a metabolic inflexibility phenotype, highlighting BAT as a critical target of PCB-126–induced toxicity and a potential contributor to systemic metabolic dysfunction.

Some limitations should be acknowledged. Experiments were performed under standard housing conditions, without cold challenge, which may limit the assessment of maximal BAT thermogenic capacity. Mechanistic interpretation is primarily based on markers of UCP1-dependent thermogenesis and β-adrenergic signaling, whereas UCP1-independent pathways of energy dissipation (e.g., creatine or calcium cycling) were not evaluated. Tissue-specific PCB-126 quantification and comparative analysis of AhR expression across organs were not conducted, as the study was not designed for toxicokinetic or receptor-mapping purposes. In addition, direct extrapolation of the administered dose to human exposure is limited, since environmental exposure occurs at low doses over prolonged periods, leading to gradual accumulation in lipid-rich tissues. Accordingly, this model should be interpreted as representing chronic, environmentally relevant metabolic stress rather than direct human exposure. Despite these limitations, the consistent BAT-specific molecular, inflammatory, redox, and functional alterations support brown adipose tissue as a sensitive target of PCB-126–induced metabolic toxicity.

PCB-126 exposure, through AhR agonism, leads to the transcriptional repression of Prdm16, PGC-1α, Ucp1, and Cidea. This repression is followed by downregulation of Pparα and AMPK2, which are essential for lipid oxidation and energy signaling. In parallel, Vegfa expression is reduced, impairing vascularization and oxygenation of the tissue. These alterations are accompanied by increased levels of TNF-α and IL-6, promoting inflammation and β-adrenergic desensitization. Therefore, Adrb3 expression is reduced, leading to impaired thermogenesis. This cascade culminates in oxidative stress, characterized by elevated MDA and NOx together with reduced activities of SOD, GPx, and catalase.

iBAT plays a pivotal role in systemic glucose and lipid homeostasis by acting as a major site of insulin-independent glucose uptake and fatty acid oxidation^35–37. Therefore, disruption of iBAT thermogenic capacity has direct consequences for whole-body metabolic regulation. In the present study, PCB-126–induced suppression of UCP1, PRDM16, PGC-1α, β3-adrenergic signaling, AMPK2, and VEGFA, together with increased inflammation and oxidative stress, indicates a profound loss of iBAT metabolic competence. This impairment likely reduces substrate clearance and energy dissipation, favoring visceral fat accumulation and contributing to glucose intolerance and insulin resistance. Notably, these systemic alterations occurred independently of changes in food intake or body weight, highlighting iBAT dysfunction as a key mechanistic driver of PCB-126–induced metabolic inflexibility.

Future studies building on the present findings should therefore combine chronic PCB-126 exposure with targeted BAT-inactivation strategies, including UCP1-dependent models and controlled thermoneutral conditions, to directly assess the contribution of BAT dysfunction to systemic metabolic alterations. Such designs will be essential to disentangle BAT-dependent from BAT-independent mechanisms and to clarify the extent to which impaired thermogenic capacity mediates the metabolic consequences of PCB-126 exposure. The present work provides a necessary foundation for these mechanistic extensions by establishing BAT as a sensitive metabolic target of chronic PCB-126 exposure under physiological housing conditions.

Methods

Animals and experimental groups

Male C57BL/6 mice (3 months of age) were housed under controlled environmental conditions, including a 12 h light/dark cycle, temperature of 21 ± 2 °C, and relative humidity of 60 ± 10%, with ad libitum access to standard chow and water. All procedures involving animals were approved by the Animal Ethics Committee of the State University of Rio de Janeiro (protocol CEUA/013) and were performed in strict accordance with all relevant institutional, national, and international guidelines and regulations for the care and use of laboratory animals. All protocols complied with the Guide for the Care and Use of Laboratory Animals (National Research Council, USA) and the Brazilian National Council for Animal Experimentation Control (CONCEA) guidelines. Every effort was made to reduce the number of animals used and to minimize suffering. We confirm that the study is reported in accordance with the ARRIVE guidelines³⁸. Animals were randomly allocated into two experimental groups (n = 10/group) to receive either PCB 126 (5 µmol/kg body weight, dissolved in corn oil) or vehicle (corn oil alone). Treatments were administered by intragastric gavage at weeks 2, 4, 6, and 8 of the experimental protocol (Fig. 1). The exposure protocol and dose selection were based on previous experimental studies demonstrating that intermittent administration of PCB-126 at 5 µmol/kg body weight is sufficient to activate aryl hydrocarbon receptor (AhR) signaling and induce metabolic, inflammatory, and oxidative alterations without causing overt systemic toxicity^4,13,17. The intermittent gavage regimen was designed to mimic chronic environmental exposure to a persistent, bioaccumulative compound rather than an acute toxic insult. This approach allows the investigation of subclinical metabolic dysfunction under conditions of preserved food intake and body weight.

All animals were fed a normocaloric, normolipidic standard diet (14% energy from protein, 10% from fat, and 76% from carbohydrates; total energy value of 15 kJ/g). The diet was manufactured by PragSoluções (Jaú, São Paulo, Brazil) in accordance with the recommendations of the American Institute of Nutrition (AIN-93 M)³⁹. Food intake and body mass were recorded weekly. After 10 weeks of experimentation, animals were subjected to a 6 h fasting period and subsequently anesthetized with an intraperitoneal injection of sodium thiopental (60 mg/kg body mass) combined with lidocaine (2%; 10 mg). Blood was collected by cardiac puncture into heparinized syringes, centrifuged at 3000 × g for 15 min at 4 °C, and plasma was separated and stored at − 20 °C until biochemical analyses. iBATs were excised, weighed, snap-frozen in liquid nitrogen, and stored at − 80 °C or fixed in formalin for further biochemical and histological assessments.

Oral glucose tolerance test

The oral glucose tolerance test (OGTT) was performed in the ninth week of treatment, that is, one week before euthanasia, using a 25% glucose solution (1.0 g/kg). The solution was administered via oral gavage after a fasting period of 6 h. Blood samples were collected from the caudal vein. Blood glucose was measured using an Accu-Check glucometer (Roche^®, SP, Brazil) at 0, 15, 30, 60, and 120 min after glucose administration.

Infrared thermography

Interscapular brown adipose tissue–associated temperature was assessed by infrared thermography. Mice were gently restrained and imaged using an infrared thermal camera (FLIR Systems, USA) under controlled ambient temperature conditions (22 ± 1 °C). Thermal images were acquired focusing on the interscapular region corresponding to the anatomical location of the iBAT depot. The mean surface temperature of the region of interest was quantified using the manufacturer’s software. Measurements were performed one week before euthanasia (n = 5).

Plasma biochemical analyses

Total cholesterol, triacylglycerol, fasting glucose, plasma alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were analyzed from a plasma sample of C57BL/6 mice using enzymatic colorimetric tests, following the manufacturer’s instructions (Bioclin^®, MG, Brazil). Insulin (#EZRMI-13-K, Millipore, MO), interleukin-6 (IL-6; #BMS603-2, Invitrogen, CA, USA), and tumor necrosis factor-alpha (TNF-α; #88-7324-88 Invitrogen, CA, USA) were measured using the ELISA test, following the manufacturer’s instructions. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the formula: HOMA-IR = glycemia concentration (mmol) x insulin concentration (IU/ml) ÷ 22.5.

Histological and stereological evaluation of interscapular brown adipose tissue (iBAT)

Formalin-fixed iBAT samples were embedded in Paraplast Plus (Sigma-Aldrich, St. Louis, MO, USA), sectioned at 5 μm, and stained with hematoxylin and eosin. Digital micrographs of random, non-consecutive fields were captured using an Olympus BX51 microscope equipped with an Infinity 15c camera (Lumenera Co., Ottawa, ON, Canada). Five animals per group were analyzed, with ten images per animal. Stereological analyses were performed using Image-Pro Plus software (version 7.0, Media Cybernetics, Silver Spring, MD, USA). The numerical density of nuclear profiles (QA [nuclei]) was estimated as previously described⁴⁰. Images were further processed with STEPanizer (www.stepanizer.com), and the number of nuclei within the defined test area (excluding those intersecting forbidden lines) was counted and normalized to the tissue area (µm²).

Immunofluorescence of UCP1 in interscapular brown adipose tissue

For immunofluorescence, iBAT sections (5 µm) were subjected to antigen retrieval and blocking of nonspecific binding with 5% bovine serum albumin and 2% glycine. They were subsequently incubated overnight with primary antibody: anti-UCP1 (PA025554ESR2HU, Cusabio, Houston, TX, USA; 1:50). Secondary antibodies conjugated to fluorophores (Alexa Fluor 488 and 546, Invitrogen, CA, USA; 1:100) were applied, followed by nuclear counterstaining with 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA). Slides were mounted with ProLong Gold Antifade (Molecular Probes, Invitrogen, Carlsbad, CA, USA), and digital images were acquired using a fluorescence microscope (EVOSfl, AMG, Advanced Microscopy Group, WA, USA).

Oxidative stress parameters

Malondialdehyde (MDA)

iBAT was homogenized in cold 1.15% KCl (1 mL per 100 mg of tissue). Aliquots of 250 µL of each homogenate were mixed with 1% phosphoric acid and 0.6% thiobarbituric acid (aqueous solution). The mixture was vortexed and heated in a boiling water bath for 45 min, then rapidly cooled on ice. Next, 4 mL of n-butanol was added, and the organic phase was separated by centrifugation at 1,200 rpm for 15 min. Absorbance was measured at 535 nm and 520 nm, and the difference between the two readings was used to calculate TBARS levels. Results were expressed as nanomoles of TBA-reactive substances per milligram of tissue (nmol/mg).

Nitrite

Nitrite (NO2) was determined using the reaction principle is based on the formation of azo compounds. Briefly, 50 µl of each sample were added in duplicate to a 96-well plate. Subsequently, 50 µl of the solution for use formed by 0.1 g of N- (1-Naphthyl) Ethyl-Enediamine (naphthyl ethylenediamine) + 1 g sulfanilamide + 90 mL of distilled water + 2.5 mL of phosphoric acid was added. After an interval of 10 min, the samples were shaken for 10 s and measured using spectrophotometry (540 nm).

Superoxide dismutase

Enzyme activity of superoxide dismutase (SOD) was determined based on the measurement of the adrenochrome concentration resulting from the oxidation of noradrenaline by the superoxide anion. The amounts of 0.5, 1, and 1.5 µL of the sample were used. The samples were incubated with 180 µL of the base solution, formed from 10 mL of the glycine solution (0.75 g of glycine for each 200 mL of distilled water at pH 10.2) + 111 µL of the catalase solution (0.2234 g of catalase for each mL of distilled water). After placing the sample in the base solution, 4 µL of noradrenaline (19 mg/mL of distilled water) was added. The adenochrome concentration was measured using spectrophotometry at 490 nm at intervals of one 180 s.

Glutathione peroxidase

The glutathione peroxidase (GPx) activity was determined from the rate of NADPH decay by spectrophotometry (340 nm). The samples were incubated for 10 min with 1450 µL of the base solution, composed of phosphate buffer (20 mL—0.078 M) + reduced glutathione (4 mL—12 mg diluted in MilliQ water) + glutathione reductase (4–20 µl diluted in phosphate buffer 0.078 M) and sodium azide (1 mL—0.065 mg diluted in MilliQ water). After the incubation time, 200 µL of NADPH was added. After 3 min, 200 µL of hydrogen peroxide (3%) was added and the reading was performed with an interval of 10 s and a duration of 300 s.

Catalase

The enzymatic activity of catalase (CAT) was determined using spectrophotometry. Briefly, 20 µL of the sample were used in separate cubettes (quartz). The samples were incubated in 1980 µL of buffer solution formulated as follows: 25 mL of phosphate buffer for each 40 µL of hydrogen peroxide (0.16%). The concentration of hydrogen peroxide was evaluated for 60 s by spectrophotometry (240 nm) at intervals of 30 s.

Quantitative reverse transcription PCR (qRT-PCR)

Total RNA was isolated from iBAT using Trizol reagent (Sigma-Aldrich, Switzerland), and RNA concentration was determined spectrophotometrically (BioDrop µLITE, BioDrop, UK). Complementary DNA (cDNA) was synthesized using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems, Life Technologies, USA). Gene expression analysis was performed by real-time PCR (7500 Fast Real-Time PCR System, Applied Biosystems, USA) using TaqMan Gene Expression Assays (Applied Biosystems, USA) specific for each target gene. Relative mRNA expression levels were calculated using the 2^(-ΔΔCt) method⁴¹. The following genes were assessed: Il-6 (Mm00446190_m1), Tnf-α (Mm01717107_m1), Pparγ (Mm00440940_m1), Prdm16 (Mm00712556_m1), Pgc-1α (Mm01208835_m1), Ucp-1 (Mm01244861_m1), Adrβ3 (Mm02601819_g1), Gadd45b (Mm00435123_m1), Cidea (Mm00432554_m1), Ampk2 (Mm01264788_m1) and Vegfa (Mm00437306_m1).

Statistical analysis

Results are expressed as mean ± standard deviation. Statistical analyses and graph construction were performed using GraphPad Prism version 10.2 (GraphPad Software, La Jolla, CA, USA). Normality and homogeneity of variance were tested using the Bartlett test. For the oral glucose tolerance test, the area under the curve (AUC) was calculated using the trapezoidal method based on blood glucose measurements obtained at 0, 15, 30, 60, and 120 min following glucose administration. AUC values were used as an integrated index of glucose tolerance. Comparisons between groups were conducted using the Student’s t-test, with statistical significance set at p < 0.05.

Author contributions

T.C. Peixoto, C.S. Miranda and (A) Teixeira: Conceptualization, Methodology, Formal analysis, Visualization, Investigation, Writing - review & editing. F.T. Quitete and (B) C. Martins: Conceptualization and Methodology. A.V.S. Teixeira and E.B. Monteiro: Methodology and investigation. D.B Mucci, A.C Resende and F.F. Martins: Methodology and analysis. J.B. Daleprane: Conceptualization, validation, visualization, Writing - review & editing.

Funding

Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro-FAPERJ E-26/211.193/202; E-26/201.234/2022; E-26/210.332/2022 granted to Dr. Julio Daleprane; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001CAPES; Conselho Nacional de Desenvolvimento Científico e Tecnológico -CNPq # 404446/2021-3.

Data availability

All data generated or analyzed during this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.