Highlights
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• High-sucrose diet (HSD) induces insulin resistance (IR).
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• HSD promotes hypertrophy and oxidative damage in interscapular brown adipose tissue (iBAT).
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• Small litter size (SL) attenuates IR in BAT by upregulating p-AKT (Ser473).
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• SL decreases PGC1-α expression, resulting in UCP1 upregulation and iBAT hyperplasia.
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• SL reduces PKA activation and FFA release, improving the oxidative damage HSD-induced.
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• SL-related iBAT activation mitigates HSD-induced IR.
Introduction
Metabolic imprinting refers to an adaptive process triggered by early-life environmental exposures, such as nutritional experiences, that induce long-lasting alterations in energy homeostasis, insulin sensitivity, and adipose tissue function.Reference Levin1,Reference Bonet, Ribot and Sánchez2 These programming effects can modulate susceptibility to or protection against chronic metabolic disorders when challenged later in life, such as during HSD exposure.Reference Waterland and Garza3,Reference Oben, Mouralidarane and Samuelsson4 Nutrition plays a crucial role in shaping the epigenome throughout critical windows of development, such as the neonatal period, where it can influence gene expression and long-term metabolic outcomes.Reference Jiménez-Chillarón, Díaz and Martínez5 In rats, the neonatal period spans from postnatal day (P0 to P7), followed by the postnatal period (suckling), which extends until weaning (P8 to P21). These stages represent critical windows for metabolic imprinting.Reference Semple, Blomgren, Gimlin, Ferriero and Noble-Haeusslein6,Reference Sengupta7 During this period, early-life environmental and nutritional factors can have lasting effects on metabolic programming, influencing long-term physiological adaptations. Such early-life experiences may underlie the early onset or delayed manifestation of metabolic diseases throughout life, characterizing the concept of the Developmental Origins of Health and Disease (DOHaD).Reference Penkler, Hanson, Biesma and Müller8
In rodents, the activation of iBAT occurs primarily through sympathetic nervous system stimulation in response to cold, physical activity, or specific dietary factors. This activation involves β3-adrenergic receptor signaling, leading to increased expression of uncoupling protein 1 (UCP1), which promotes mitochondrial fatty acid oxidation and alters cellular energy handling.Reference Lettieri-Barbato9 Although iBAT depots are less prominent in adulthood, this tissue remains metabolically active and plays a significant role in regulating glucose homeostasis and systemic energy balance.Reference Schnaider and Borges10 Its responsiveness to environmental and nutritional stimuli during early development highlights its relevance in the context of metabolic programming.
Emerging evidence indicates that short-term cold exposure can enhance energy expenditure and increase iBAT activity in adults, contributing to improved metabolic flexibility and representing a potential target for obesity management.Reference Huo, Song and Yin11 However, while acute activation of iBAT may transiently buffer energy surplus, chronic exposure to highly palatable diets, such as HSD, can elicit maladaptive responses, including ectopic lipid accumulation and IR. Notably, HSD has been shown to increase both the metabolic and proliferative activity of iBAT, increasing circulating triacylglycerol (TAG) and free fatty acids (FFA) levels.Reference Saito, Matsushita, Yoneshiro and Okamatsu-Ogura12 These changes are accompanied by increased tissue mass and upregulation of UCP1, reflecting a compensatory adaptation aimed at preserving energy balance.Reference Lettieri-Barbato9 Despite the initial benefits of such responses, the chronic elevation of circulating lipids may exacerbate metabolic dysfunction, promoting IR and increasing the risk of metabolic diseases. This paradoxical role highlights the complexity of iBAT activation in response to dietary stimuli, acting both as a protective mechanism and a potential contributor to metabolic imbalance. Importantly, iBAT plays a key role in modulating metabolic systemic metabolism, as brown adipocytes are capable of oxidizing intracellular FFA or circulating glucose, thereby influencing whole-body insulin sensitivity and energy homeostasis.Reference Saito, Matsushita, Yoneshiro and Okamatsu-Ogura12
In animal models, a SL during the postnatal period promotes neonatal overfeeding and has been used to better understand the short- and long-term metabolic consequences;Reference Spencer13,Reference Habbout, Li, Rochette and Vergely14 however, this is a controversial experimental model. Some authors suggest that overfeeding may worsen disease onset due to being overweight in small litters after weaning,Reference Xiao, Williams and Grayson15,Reference Isganaitis, Woo and Ma16 which may induce hyperphagia, overweight, and hyperinsulinemia throughout life.Reference Spencer13,Reference Habbout, Li, Rochette and Vergely14 On the other hand, others indicate that the impact on adverse metabolic changes may be temporary, or even beneficial, due to metabolic plasticity, allowing SL rats to better adapt to variations in diet throughout life. This suggests that long-term body mass gain is mild and not always associated with hyperphagia or diabetes indices in adulthood.Reference Stefanidis and Spencer17,Reference Cai, Dinan and Barwood18 However, although the SL model gives valuable insights into neonatal overfeeding metabolism, the divergent perspectives on its long-term effects highlight the need for further research to clarify the complex interplay between early nutritional experiences and lifelong metabolic health.
Notably, an HSD may promote IRReference de Queiroz, Coimbra and Ferreira19,Reference de Queiroz, Evangelista and Guerra-Sá20 and iBAT activation by increasing iBAT size and UCP1 expression.Reference de Queiroz, Rodovalho and Guimarães21 In contrast, the SL may result in neonatal overfeeding, which reduces the thermogenic activity in adult rats, decreasing iBAT β3-adrenergic receptor and UCP1 expressions.Reference Isganaitis, Woo and Ma16 Moreover, the impairment of the insulin signaling pathway during IR culminates in a decrease in protein kinase B (AKT) phosphorylation at serine 473 (p-AKT[Ser473]), which blocks peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) phosphorylation and downregulates the UCP1 expression.Reference Saltiel22 Thus, there is a positive correlation between p-AKT (Ser 473), PGC1-α, and UCP1 expression in iBAT, which activates iBAT thermogenesis and mitigates IR.Reference Ortega-Molina, Efeyan and Lopez-Guadamillas23
The increase in mitochondrial β-oxidation through β3-adrenergic signaling stimulation during IR HSD-induced results in an increased reactive oxygen species (ROS) production.Reference Corbi, Conti and Russomanno24 Furthermore, the SL model is related to changes in the post-weaning redox process, predisposing adults to IR. However, after being fed a high-fat diet, small-litter rats are no longer susceptible to these short-term metabolic adverse effects.Reference Cai, Dinan and Barwood18 iBAT is a highly oxidative tissue, rich in mitochondria, and its activity relies on sustained fatty acid oxidation and elevated oxygen consumption, both of which contribute to increased ROS generation.Reference Cannon and Nedergaard25,Reference Chouchani, Kazak and Jedrychowski26 While ROS play essential roles in cellular signaling, their excessive production may disrupt redox homeostasis and impair iBAT function. HSD has been associated with oxidative stress and mitochondrial dysfunction in peripheral tissues such as the liver and skeletal muscle.Reference Lima-Leopoldo, Leopoldo and Silva27,Reference de Queiroz, Honorato-Sampaio and Rossoni Junior28,Reference Rodrigues, Rosa and Medeiros29 However, the specific effects of HSD on redox balance in iBAT remain poorly understood. Therefore, investigating how HSD-driven metabolic imprinting affects iBAT activation and redox adaptations is essential for understanding its long-term impact on adult metabolism.
We have previously demonstrated that rats fed an HSD develop IRReference de Queiroz, Coimbra and Ferreira19,Reference de Queiroz, Evangelista and Guerra-Sá20 and exhibit iBAT activation, characterized by increasing tissue size, mRNA, and UCP1 protein expression after eight weeks.Reference de Queiroz, Rodovalho and Guimarães21 However, this activation did not prevent fat gain. Given that the SL and post-weaning HSD may predispose iBAT to permanent metabolic changes, we aimed to investigate whether the metabolic imprinting resulting from the SL could mitigate the development of HSD-induced IR through iBAT activation in young rats. Additionally, we investigated whether this metabolic imprinting influenced redox processes in brown adipocytes affected by HSD-induced IR. We hypothesized that the metabolic imprinting would enhance iBAT activation and modulate redox balance, thereby alleviating HSD-induced IR. This study represents the first comprehensive examination of the molecular mechanism underlying iBAT activation and its potential role in alleviating HSD-induced IR in a SL model.
Materials and methods
Animals and diets
The animals were divided into two dietary groups: the standard diet (STD) group, fed a Nuvilab® commercial chow (Nuvital-CR, Colombo, Brazil), while the high-sucrose (HSD, 30% sucrose) group was fed a “palatable” diet based on condensed milk (40.44%) and sugar (8.57%), as previously published.Reference Oliveira, Fernandes and Sousa30
The centesimal compositions of the diets are presented in Table 1. Body mass was measured weekly, and food intake was monitored three times a week. Energy intake was calculated by multiplying the weekly food intake by the energy densities of the STD (310 kcal) and HSD (313 kcal).
Table 1. Ingredient and nutrient composition of diets fed to rats for an 8-week period
The standard diet consists solely of Nuvilab® commercial chow (Nuvital-CR, Colombo, Brazil). HSD diet comprises 40.44% Nuvilab® commercial chow, 40.44% Nestlé Moça condensed milk, 8.57% sugar, and 10.53% water, as previously describedReference Oliveira, Fernandes and Sousa30.
g: grams; NA, not applicable; kcal, kilocalorie; kJ, kilojoules.
a Adapted byReference Oliveira, Fernandes and Sousa30.
b based on the manufacturer’s information.
Experimental protocol
This study was approved by the Ethics Committee for the Use of Animals at the Federal University of Ouro Preto (UFOP) (protocol number 2245040518). The effect of litter size was determined as previously described.Reference Spencer and Tilbrook31 After birth, male Wistar rats (n = 48) were randomly assigned to either the control litter (8 pups/dam; CL) or small litter (4 pups/dam; SL). Post-weaning (21 days), the animals were redistributed into four groups: (i) animals fed STD (Nuvilab® commercial chow) from CL (n = 12) or (iii) SL (n = 12); (ii) animals fed a HSD (30% sucrose) from CL (n = 12) or SL (n = 12). The animals were housed at a temperature of 24 ± 2°C, with a 12-hour light/dark cycle, and they had ad libitum access to water and food.
Euthanasia
At the end of the eight-week feeding period, the non-fasted animals were anesthetized with isoflurane (Isoforine®, São Paulo, Brazil) and euthanized by decapitation. Isoflurane, a short-acting anesthetic (lasting less than 2 minutes), was used per ethical guidelines and did not affect the Rat Grimace Scale (RGS) for pain assessment.Reference Miller, Golledge and Leach32 Its brief use before euthanasia was expected to minimally impact outcomes.Reference Maharani, Fadlyah and Setyaningrum33 All animals underwent the same procedure for consistency. After euthanasia, serum, white adipose tissue (WAT) (retroperitoneal, epididymal, and inguinal), and iBAT were collected, weighed, and stored at -80°C for analysis. All analyses performed on iBAT were conducted using samples collected under fed-state conditions to better reflect the tissue’s metabolic activity in response to nutritional stimuli.Reference Kersten34
Feeding efficiency and murinometric analyses
During the eight-week period, the rats weighed to calculate several metrics: the coefficient of feeding efficiency (CFE) [(final weight (g) / initial weight (g)) / total food intake (g)], weight gain per caloric consumption (WGCC) [(final body weight (g) — initial body weight (g)) / kcal ingested],Reference Nery, Pinheiro and Muniz35 the adiposity index (AI) [100 × (sum of fat pad weights) / body weight], and the Lee Index (LI) [3√Weight (g) / naso-anal length (cm)]. iBAT weight was expressed as a relative weight: [iBAT weight (g) / body weight (g)) × 100].Reference de Queiroz, Rodovalho and Guimarães21
Serum parameters and intraperitoneal glucose tolerance test (ipGTT)
Serum total cholesterol, high-density lipoprotein (HDL), TAG, and glucose were measured using Labtest® kits (Labtest Diagnostica SA, Minas Gerais, Brazil) in an Epoch multi-detection microplate reader (BioTek®, Washington, WA). Serum insulin levels were assayed with a rat insulin ELISA kit (Cat # EZRMI-13K; Merck Millipore, Burlington, MA). An intraperitoneal glucose tolerance test (ipGTT) was performed in fasted rats (6 h). Glucose levels in tail blood samples were monitored at 0, 30, 60, 90, and 120 min post-administration using an Accu-Check glucometer (Accu-Chek Active, Roche Diagnostics Corp., Hague Road, IN), following a published protocol.Reference Santos, Fernandes and Mario36 Data are presented as the area under the curve (AUC). IR was assessed using the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) formula: (fasting serum insulin × plasma glucose) / 22.5.Reference Matthews, Hosker, Rudenski, Naylor, Treacher and Turner37 The quantitative insulin sensitivity check index (QUICKI) was calculated as: 1 / [log(fasting insulin mIU/mL) + log(fasting glucose mg/dL)].Reference Chen, Sullivan and Quon38
Brown adipocyte size and lipid content
iBAT fat pad sections were fixed in 80% methanol-20% DMSO solution (Methanol, Alphatec, Rio de Janeiro, Brazil; DMSO, Labsynth, São Paulo, Brazil) for 72 hours, embedded in paraffin, sliced to 5 μm thickness with a microtome, and stained with hematoxylin-eosin. Sections were visualized microscopically at 40× magnification. The images were digitized and analyzed with the Leica Qwin V3 software (Leica Microsystems, Wetzlar, Germany). The number and area of brown adipocytes were quantified in the iBAT, covering a total area of 2592 × 1944 µmReference Bonet, Ribot and Sánchez2,Reference de Queiroz, Coimbra and Ferreira19 Brown adipocytes were examined blindly across experimental groups.
Lipid content was measured using the Folch method,Reference Folch, Lees and Stanley39 and expressed as grams of lipids per relative iBAT weight. Extracted lipids were resuspended in isopropyl alcohol (Alphatec) for TAG levels analysis with a Triglycerides Liquiform kit (Labtest), following the manufacturer’s instructions.
Western blot analysis
Protein levels of of p-AKT(Ser 473), total PGC1-α, UCP1, and p-PKA substrate proteins were determined using western blotting with specific rabbit polyclonal antibodies: anti-AKT (#SAB4500799), anti-phospho-AKT (pSer473) (#SAB4504331) (Sigma-Aldrich, San Louis, MO), anti-phospho (Ser/Thr) PKA substrates (#9621), anti-PGC1-α (#4259) (Cell Signaling Technology, Danvers, MA)), and anti-UCP1 (AB1426, Merck Millipore). Fifty milligrams of iBAT were homogenized in a RIPA lysis buffer, centrifuged (12,000 × g at 4°C for 40 min), and total proteins were quantified using the Lowry method.Reference Lowry, Rosebrough, Farr and Randall40 Protein samples (60 μg) were separated by SDS-PAGE (10% for p-AKT(Ser 473) and total PGC1-α, 16% for UCP1, and 8% for p-PKA substrates) and transferred to nitrocellulose membranes (BioRad®, Hercules, CA). After overnight incubation with primary antibodies (1:1000 for anti-AKT, anti-p-AKT, anti-UCP1, anti-p-PKA substrates, and anti-α-tubulin; 1:500 for anti-PGC1-α), secondary antibodies (1:5000 anti-rabbit, #4414S, Cell Signaling) were applied for detection and visualized by the ECL system (Sigma-Aldrich). Protein expression was normalized to total protein levels, determined by Ponceau S staining of the PVDF membrane post-transfer. Band intensities were adjusted according to the total membrane signal to ensure accurate comparison across samples.Reference Sander, Wallace, Plouse, Tiwari and Gomes41 Relative protein expression was analyzed using ImageJ software (version 1.51k, National Institutes of Health, accessed at http://rsb.info.nih.gov/ij/).
Free fatty acids quantification
After 8 weeks, the levels of FFA in the iBAT were measured using a commercial kit (#MAK044, Sigma-Aldrich) according to the manufacturer’s protocol. Subsequently, absorbance was read at 570 nm using an Epoch multi-detection microplate reader (Biotek®).
Measurement of oxidative stress-related indicators
iBAT homogenate was prepared with 100 mM EDTA (pH 7.8) (1:10) and a 1:100 protease inhibitor cocktail (PIC #P8340; Sigma-Aldrich). Total protein was measured using the Lowry method.Reference Lowry, Rosebrough, Farr and Randall40 Antioxidant profiles were assessed by measuring superoxide dismutase (SOD) activity, which produces a purplish color with MTT [3-(4,5-dimethyl-thiazol 2-yl) 2,5-diphenyl tetrazolium bromide], due to pyrogallic acid oxidation, read at 570 nm.Reference Madesh and Balasubramanian42 Catalase (CAT) activity was determined by hydrogen peroxide decay at 240 nm.Reference Aebi43 Sulfhydryl groups were quantified by their reaction with DTNB (5,5’-dithio-bis-(2-nitrobenzoic acid)), read at 412 nm.Reference Sedlak and Lindsay44 Carbonylated proteins were analyzed using DNPH (2,4-dinitrophenylhydrazine), yielding a hydrazone read at 370 nm.Reference Levine, Williams, Stadtman and Shacter45 Lipid peroxidation was measured by malondialdehyde (MDA) levels using the thiobarbituric acid reactive substances (TBARS) method as previously described.Reference Buege and Aust46,Reference Conceição, Franco and Oliveira47,Reference Rolnik, Olas and Szablińska-Piernik48 The absorbance of the organic phase containing the pink chromogen was measured at 532 nm. Equivalents were expressed in nMol/mg protein.
Statistical analyses
The sample size was determined to ensure a power of 0.9 and a significance level (α) of 0.05, based on the minimum expected difference between treatment means. The “Lee index” was chosen for its highest variability in previous analyses, leading to a maximum estimated sample size of n = 13 per group. Data normality was assessed with the Shapiro–Wilk test. Parametric data are presented as mean ± standard deviation (SD) and illustrated using bar graphs, whereas non-parametric data are reported as median with interquartile range (IQR) and displayed using box plot. Effects of litter size and post-weaning diet were analyzed by TWO-WAY ANOVA, followed by Bonferroni post-test when interaction was observed. For comparisons of the main effects (litter size and/or post-weaning diet), post-hoc mean comparison tests (Student’s t or Mann–Whitney) were used to identify specific group differences. Outliers were excluded using the IQR criterion when applicable. Statistical analyses were conducted using GraphPad Prism software (version 9.0; GraphPad Software Inc., Irvine, CA), and differences were considered significant at P < 0.05.
Results
Murinometric and metabolic characteristics of rats in the small litter size (SL) model with a high-sucrose diet (HSD)
The murinometric and lipid profiles data are summarized in Table 2. The SL model increased postnatal weight by ∼ 10% (59.05 ± 8.32g) compared to the control litter (53.65 ± 3.03g, P <0.01). After eight weeks on an HSD, daily calorie intake was influenced by litter size (P <0.01) and post-weaning diet (P <0.001), with the HSD-SL group showing increased intake compared to the STD-SL and HSD-CL groups. The CFE and WGCC were affected solely by the post-weaning diet (P <0.05 and P <0.001, respectively). The CFE in the HSD-CL group was lower than that in the STD-CL group. Regarding WGCC, both the HSD-CL and HSD-SL groups demonstrated a decrease compared to the STD-CL and STD-SL groups, respectively. No significant effects were observed on body mass gain, NAL, or LI. However, an effect of the post-weaning diet on AI was noted (P <0.001), indicating increased adiposity in the HSD-CL and HSD-SL groups compared to their respective controls (STD-CL and STD-SL groups). Regarding HDL-cholesterol levels, we noted effects of litter size (P <0.001), post-weaning diet, and their interaction (P <0.01). Specifically, the HSD-CL group showed a 55% increase compared to the STD-CL group, while the HSD-SL group exhibited a 41% reduction compared to the HSD-CL group. Additionally, both litter size and post-weaning diet influenced serum TAG levels (P <0.05). Specifically, TAG levels increased in the HSD-CL group relative to the STD-CL, whereas a decrease was observed in the HSD-SL group compared to the HSD-CL group.
Table 2. Validation of the experimental model of small litter size (SL) and insulin resistance induced by high-sucrose diet (HSD)
a Litter size was controlled as previously described: 8 pups/dam (control) and 4 pups/dam (small litter). Data normality was tested using the Shapiro-Wilk test. Parametric data are presented as mean ± standard deviation (SD), while non-parametric data are shown as median and interquartile rage (IQR). Effects of litter size and post-weaning diet were analyzed by TWO-WAY ANOVA, followed by Bonferroni post-test when interaction was observed. For comparisons of the main effects (litter size and/or post-weaning diet), post-hoc mean comparison tests (Student’s t or Mann–Whitney) were used to identify specific group differences.(*P <0.05, **P <0.001, ***P <0.0001). a, to STD; b, to HSD; c, to litter reduction; CL: control litter; SL: small litter size; g: grams; STD: standard diet; HSD: high-sucrose diet; DI: Daily intake; kJ: kilojoule; MG: Mass gain; CFE: coefficient of feeding efficiency; WGCC: weight gain per caloric consumption; NAL; naso-anal length; LI; Lee index; AI: adiposity index; TC: Total cholesterol; HDL: high-density lipoprotein; TAG: triacylglycerol.
The effects of litter size and HSD on glucometabolic parameters were also evaluated (Fig 1). The post-weaning diet had a significant overall effect on the AUC of the ipGTT (P <0.05), however, no significant pairwise differences were detected in the post-hoc analyses (Fig. 1b). Serum glucose levels were significantly affected by the post-weaning diet (P <0.05), with a 30% increase observed in the HSD-CL group compared to the STD-CL group (Fig. 1c). For insulin levels, both litter size (P <0.05) and post-weaning diet (P <0.001) had significant effects. Specifically, serum insulin levels were 104% higher in the HSD-CL group compared to the STD-CL group. In contrast, the HSD-SL group showed a 38% reduction in serum insulin levels relative to the HSD-CL group (Fig. 1d). Similarly, the HOMA-IR index was influenced by the litter size (P <0.05) and the post-weaning diet (P <0.001), with a remarkable 165% increase in the HSD-CL compared to the STD-CL group. Interestingly, a 43% reduction in HOMA-IR was observed in the HSD-SL compared to the HSD-CL group (Fig. 1e). Regarding the QUICKI index, the post-weaning diet had a significant effect (P <0.05), with an 11% decrease in the HSD-CL group compared to the STD-CL group (Fig. 1f).
Figure 1. Effect of small litter size (SL) and high-sucrose diet (HSD) on glucometabolic parameters after 8 weeks. a) intraperitoneal glucose tolerance test (ipGTT), time-course graph with median and interquartile range (mmol/L); b) area under the curve of the ipGTT; c) serum glucose (mmol/L); d) serum insulin (pmol/L); e) homeostasis model assessment of insulin resistance (HOMA-IR); f) simple quantitative insulin sensitivity check index (QUICKI). Data normality was assessed with the Shapiro–Wilk test. Parametric data are presented as mean ± standard deviation (SD) and illustrated using bar graphs, whereas non-parametric data are reported as median with interquartile range (IQR) and displayed using box plot. Effects of litter size and post-weaning diet were analyzed by TWO-WAY ANOVA, followed by Bonferroni post-test when interaction was observed. For comparisons of the main effects (litter size and/or post-weaning diet), post-hoc mean comparison tests (Student’s t or Mann–Whitney) were used to identify specific group differences (*P < 0.05, **P < 0.001, ***P < 0.0001). CL: control litter; HOMA-IR formula: (fasting serum insulin × plasma glucose) / 22.5 37; HSD: high-sucrose diet; ipGTT: intraperitoneal glucose tolerance test; mmol/L: millimoles per liter; pmol/L: picomoles per liter; QUICKI formula: 1 / [log(fasting insulin mIU/mL) + log(fasting glucose mg/dL)] 38; SL: small litter size; STD: standard diet.
Small litter size (SL) induces hyperplasia, leading to a reduction in the relative mass of interscapular brown adipose tissue (iBAT) and alters its lipid content after eight weeks of high-sucrose diet (HSD) consumption
Morphometric analysis of the brown adipocytes area was across the four experimental groups (Fig. 2a). Our results revealed that the number of brown adipocytes decreased while the area of individual adipocytes increased due to the HSD (P <0.001). Meanwhile, the interaction between litter size and post-weaning diet also had a significant effect (P <0.001), increasing the number (Fig. 2b) and reducing the adipocyte area of brown adipocytes (Fig. 2c). Concerning iBAT relative mass, we found that litter size, post-weaning diet, and their interaction all influenced outcomes (P <0.05). Specifically, the HSD-CL group exhibited an increase in iBAT relative mass, whereas the HSD-SL group showed a decrease compared to both the STD-SL and HSD-CL groups (Fig. 2d). Regarding the percentage of iBAT lipids, an interaction effect was evident (P <0.001), with lipid content increasing in both the STD-SL and HSD-CL groups compared to the STD-CL group. Notably, the HSD-SL group demonstrated a reduction in lipid percentage compared with the HSD-CL group (Fig. 2e). For tissue TAG levels, we observed effects from litter size (P <0.05), post-weaning diet (P <0.01), and their interaction (P <0.001). The STD-SL group showed increased TAG levels (compared to the STD-CL group), while the HSD-SL group exhibited a decrease in TAG compared to the STD-SL group (Fig. 2f).
Figure 2. Effect of small litter size (SL) and high-sucrose diet (HSD) on multilocular adipocytes in interscapular brown adipose tissue (iBAT) after 8 weeks. a) histological sections of multilocular adipocytes stained with hematoxylin-eosin, with representative adipocytes indicated by black arrows (scale bar = 50 μm). b) the number of adipocytes, determined by counting nuclei in a fixed area (2592 × 1944 μmReference Bonet, Ribot and Sánchez2) using leica qwin v3.5.1 software, across 15 fields per slide. c) average area of adipocytes calculated from measurements in the same fields. d) iBAT relative mass; e) percentage of total lipids relative to iBAT mass. f) triacylglycerol (TAG) levels in iBAT. Litter size was manipulated, with control litters consisting of 8 pups per dam and small litters of 4 pups per dam. Data normality was assessed with the Shapiro–Wilk test. Parametric data are presented as mean ± standard deviation (SD) and illustrated using bar graphs, whereas non-parametric data are reported as median with interquartile range (IQR) and displayed using box plot. Effects of litter size and post-weaning diet were analyzed by TWO-WAY ANOVA, followed by Bonferroni post-test when interaction was observed. For comparisons of the main effects (litter size and/or post-weaning diet), post-hoc mean comparison tests (Student’s t or Mann–Whitney) were used to identify specific group differences (*P < 0.05, **P < 0.001, ***P < 0.0001). %: percent; HSD: high-sucrose diet; iBAT: interscapular brown adipose tissue; mg/dL: milligrams per deciliter; STD: standard diet; TAG: triacylglycerol; μmReference Bonet, Ribot and Sánchez2: square microns.
Small litter size (SL) enhances the activation of interscapular brown adipose tissue (iBAT) and modulates its response to insulin resistance (IR) induced by a high-sucrose diet (HSD), through regulating the expression of p-AKT(Ser 473), UCP1, and PGC1-α
Figure 3 represents the protein contents analyzed via western blotting. For p-AKT (Ser 473), effects of litter size (P <0.05) and the interaction between variables (P <0.001) were observed, with a 36% downregulation in the HSD-CL group compared to the STD-CL group. Conversely, the HSD-SL group showed a 66 and 110% upregulation compared to the STD-SL and the HSD-CL groups, respectively (Fig. 3a).
Figure 3. Effect of small litter size (SL) and high-sucrose diet (HSD) on the protein expression of p-AKT(Ser 473), PGC1-α and UCP1 in interscapular brown adipose tissue (iBAT) after 8 weeks. a) phosphorylation levels of p-AKT (Ser 473); b) protein content of PGC1-α; c) protein content of UCP1; d) representative blots showing the protein levels of p-AKT(Ser 473), total PGC1-α and UCP, the results were normalized by total membrane protein stained with ponceau. Litter size was manipulated, with control litters consisting of 8 pups per dam and small litters of 4 pups per dam. Data normality was assessed with the Shapiro–Wilk test. Parametric data are presented as mean ± standard deviation (SD) and illustrated using bar graphs, whereas non-parametric data are reported as median with interquartile range (IQR) and displayed using box plot. Effects of litter size and post-weaning diet were analyzed by TWO-WAY ANOVA, followed by Bonferroni post-test when interaction was observed. For comparisons of the main effects (litter size and/or post-weaning diet), post-hoc mean comparison tests (Student’s t or Mann–Whitney) were used to identify specific group differences (*P < 0.05, **P < 0.001, ***P < 0.0001). CL: control litter; HSD: high-sucrose diet; kDa: kilodalton; p-AKT[Ser473]: protein kinase B (AKT) phosphorylation at serine 473; PGC1-α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; SL: small litter size; STD: standard diet; UCP1: uncoupling protein 1.
Regarding total PGC1-α, litter size and the post-weaning diet (P <0.01) influenced protein levels, with a 42% downregulation in the STD-SL group versus the STD-CL group and a 42% upregulation in the HSD-CL group compared to the STD-CL group. The HSD-SL group exhibited a 40% decrease in total PGC1-α compared to the HSD-CL (Fig. 3b).
For UCP1, significant effects were also noted (litter size: P <0.05; post-weaning diet: P <0.001), with a 32% upregulation in the STD-SL group and a 14% increase in the HSD-CL group, relative to the STD-CL group. The HSD-SL group showed a 21% increase in UCP1 compared to the STD-SL, and SL raised levels by 9% in HSD-SL compared to the HSD-CL group (Fig. 3c). Panel D displays the blots of the p-AKT (Ser 473), total PGC1-α, and UCP1 (Fig. 3d).
Small litter size (SL) decreased PKA substrate phosphorylation and free fatty acids (FFA) levels in the interscapular brown adipose tissue (iBAT) of rats fed a high-sucrose diet (HSD)
Figure 4 illustrates PKA substrate phosphorylation (Fig. 4a), blot p-PKA (Ser/Thr) substrate (Fig 4b), and the iBAT FFAs levels (Fig. 4c) after eight weeks. For PKA substrate phosphorylation, a significant interaction effect was noted (P <0.001), with a 94% upregulation in the STD-SL group and a 135% upregulation in the HSD-CL group compared to the STD-CL group. Conversely, the HSD-SL group showed a 37 and 48% downregulation in PKA substrates phosphorylation compared to the STD-SL and HSD-CL groups, respectively (Fig. 4a). In terms of FFA levels, litter size had a significant effect (P <0.05), with a 61% decrease in the HSD-SL group compared to the HSD-CL group (Fig 4c).
Figure 4. Effect of small litter size (SL) and high-sucrose diet (HSD) on PKA and free fatty acid (FFA) substrates in interscapular brown adipose tissue (iBAT) after 8 weeks. a) levels of phospho-(Ser/Thr) PKA substrates. Densitometric quantification was performed considering the total lane signal to reflect the overall phosphorylation profile of PKA substrates. b) blot p-PKA (Ser/Thr) substrate; c) tissue levels of free fatty acids in iBAT, expressed in nmoles/μl. Litter size was controlled as previously described: 8 pups/dam (control litter) and 4 pups/dam (small litter). Data normality was assessed with the Shapiro–Wilk test. Parametric data are presented as mean ± standard deviation (SD) and illustrated using bar graphs. Effects of litter size and post-weaning diet were analyzed by TWO-WAY ANOVA, followed by Bonferroni post-test when interaction was observed. For comparisons of the main effects (litter size and/or post-weaning diet), post-hoc mean comparison tests (Student’s t or Mann–Whitney) were used to identify specific group differences.(*P < 0.05, **P < 0.001, ***P < 0.0001). CL: control litter; HSD: high-sucrose diet; kDa: kilodalton; nmoles/μL: nanomoles per microliter; PKA: protein kinase A; ser: serine; SL: small litter size; STD: standard diet; thr: threonine.
Small litter size (SL) mitigates lipid peroxidation measured by MDA levels in interscapular brown adipose tissue (iBAT) induced by a high-sucrose diet (HSD)
Figure 5 presents results from analyses of SOD, CAT, sulfhydryl groups, carbonylated proteins, and MDA levels. For SOD activity, a significant effect of post-weaning diet was observed (P <0.01), with increases of 59% in the HSD-CL group and 63% in the HSD-SL group compared to the STD groups (Fig. 5a). CAT activity showed significant effects from litter size (P <0.05), the post-weaning diet (P <0.001), and their interaction (P <0.01), with 19% decrease in the STD-SL group and a 275% increase in the HSD-SL group, compared to the respective STD group (Fig. 5b). Sulfhydryl groups increased by 93% in the HSD-CL group when compared to the STD-CL group reflecting effects of both litter size (P <0.05) and the post-weaning diet (P <0.01) (Fig. 5c). In terms of oxidative damage markers, the carbonylated protein levels were affected by the post-weaning diet (P <0.05), with an increase of 78% in the HSD-CL group compared to the STD-CL group (Fig. 5d). Lipid peroxidation measured by MDA levels using the TBARS analysis revealed significant effects from the post-weaning diet (P <0.001) and the interaction (P <0.01), with reduction of 88 and 78% in the HSD-SL group compared to the STD-SL and HSD-CL groups, respectively (Fig. 5e).
Figure 5. Effect of small litter size (SL) model and high-sucrose diet (HSD) on the antioxidant profile and oxidative damage markers in interscapular brown adipose tissue (iBAT) after 8 weeks. a) superoxide dismutase (SOD) activity, expressed in U/mg of total proteins; b) catalase (CAT) activity, expressed in U/mg of proteins; c) sulfhydryl groups, measured in nMol/mg of total proteins. d) carbonylated proteins, quantified in nMol/mg of total proteins; e) malondialdehyde, expressed in nMol/mg of total proteins. Litter size was controlled as previously described: 8 pups/dam (control litter) and 4 pups/dam (small litter). Data normality was assessed with the Shapiro–Wilk test. Parametric data are presented as mean ± standard deviation (SD) and illustrated using bar graphs, whereas non-parametric data are reported as median with interquartile range (IQR) and displayed using box plot. Effects of litter size and post-weaning diet were analyzed by TWO-WAY ANOVA, followed by Bonferroni post-test when interaction was observed. For comparisons of the main effects (litter size and/or post-weaning diet), post-hoc mean comparison tests (Student’s t or Mann–Whitney) were used to identify specific group differences.(*P < 0.05, **P < 0.001, ***P < 0.0001). HSD: high-sucrose diet; mg: milligram; nmol/mg: nanomoles per milligram; STD: standard diet; U: unit.
Discussion
This study is the first to suggest that metabolic imprinting induced by the SL model activates iBAT and mitigates IR induced by an HSD through the upregulation of p-AKT (Ser473) and PGC1-α signaling in the tissue. Additionally, the SL model was associated with enhanced UCP1 expression and downregulation in PKA substrates phosphorylation, followed by lower levels of FFA and oxidative damage HSD-induced after eight weeks. These findings are illustrated in the following graphical abstract (Fig. 6).
Figure 6. Graphical abstract. An eight-week high-sucrose diet (HSD) induced metabolic damage, characterized by insulin resistance (IR) and hypertrophy of interscapular brown adipose tissue (iBAT). Insulin signaling in brown adipocytes was compromised, with reduced p-AKT (Ser473), leading to tissue insulin resistance. The small litter size (SL) model promotes metabolic imprinting and activates iBAT through the p-AKT (Ser473) and PGC1-α signaling pathways. This results in increased UCP1 expression and reduced fatty acid oxidation, while also mitigating oxidative damage induced by a high-sucrose diet (HSD) and downregulating PKA signaling. Although our results challenge the existing literature, we propose that the metabolic plasticity associated with the SL model enables rats to adapt to dietary variations and may offer protection against HSD-induced IR in adulthood. AC: adenylyl cyclase; AI: adiposity index; AKT: protein kinase B; AMPc: cyclic adenosine monophosphate; ATP: Adenosine triphosphate; CAT: catalase; DE: diet effect; FFA: free fatty acids; GLUT-4: glucose transporter type 4; GS: glycogen synthase; HOMA-IR: homeostasis model assessment of insulin resistance; HSD: high-sucrose diet; HSL: hormone-sensitive lipase; iBAT: interscapular brown adipose tissue; IE: interaction effect; insR: insulin receptor; IR: insulin resistance; IRS: insulin substrate receptor; mTORC2: Mammalian target of rapamycin complex 2; MDA: malondialdehyde; NE: norepinephrine; P: phosphate; PDK1: 3-phosphoinositide-dependent protein Kinase1; PGC1-α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PIP2: phosphatidylinositol 4,5-bisphosphate; PIP3: phosphatidylinositol (3,4,5)-trisphosphate; PI3K: phosphoinositide 3-kinase; PKA: protein kinase A; ROS: reactive oxygen species; ser: serine; SLE: small litter size effect; SL: small litter size; SG: sulfhydryl groups; SOD: superoxide dismutase; TAG: triacylglycerol; thr: threonine; TLR4: toll-like receptor 4; tyr: tyrosine; UCP1: uncoupling protein 1; β3: β3-adrenergic receptor; GLU: glucose.
Our SL model aligns with previously described methodologiesReference Spencer13 and effectively induces postnatal overweight due to high milk intake, leading to increased body mass post-weaning.Reference Spencer and Tilbrook31 This postnatal overfeeding associated with reduced litter size is commonly linked to early hypothalamic malprogramming, resulting in persistent central hyperleptinemia, IR, and heightened orexigenic signals.Reference Dąbrowska and Mirabegron49,Reference Conceição, Moura and Trevenzoli50 These factors may contribute to hyperphagia, overweight, and hyperinsulinemia throughout life, as noted by some researchers.Reference Spencer13,Reference Habbout, Li, Rochette and Vergely14 However, other studies indicate that long-term weight gain is not always correlated with hyperphagia or diabetes in adulthood,Reference Stefanidis and Spencer17,Reference Cai, Dinan and Barwood18 adding to the controversy surrounding this model. Considering this, our results support the hypothesis that SL fosters metabolic plasticity, potentially leading to improved IR after exposure to an HSD. The potential mechanisms underlying this effect will be explored in detail below.
According to our results, HSD-induced IR, hypertrophy, and oxidative damage in iBAT. Notably, HSD increases body fat and TAG levels, contributing to the development of IR through the upregulation of metabolic pathways that regulate fatty acid and TAG production, thereby increasing adiposity Reference Margareto, Marti and Martínez51 and exacerbating IR. It’s important to note that, despite the lower levels of protein and micronutrients in the HSD compared to the STD, no negative effects were observed in the animals throughout the 8-week experiment. All groups exhibited satisfactory body weight gain and growth, as indicated by the absence of significant differences in weight gain and naso-anal length.
Scientific evidence links IR to oxidative stress caused by high levels of glucose, insulin, and TAG, alongside inadequate antioxidant defenses,Reference Gjorgjieva, Mithieux and Rajas52 stemming from excessive ROS production.Reference Dornas, Cardoso and Silva53 It has been proposed that HSD may stimulate β3-adrenergic signaling, promoting iBAT activation and increasing its size as a compensatory mechanism to counteract diet-induced weight gain and prevent obesity.Reference Margareto, Marti and Martínez51 However, the hypertrophy observed in iBAT – characterized by an increase in adipocyte area, relative mass, and lipid content – indicates tissue dysfunction and a potential phenotypic shift. At this stage, iBAT starts to resemble WAT in a process called “whitening”, which results in greater fat storage within brown adipocytes due to less antioxidant protection during the induction of effective thermogenesis and mitochondrial ROS overproduction.Reference Ziqubu, Dludla and Mthembu54 This oxidative stress causes oxidation of cell membrane proteins and lipids, which produce by-products like carbonylated protein and MDA. The activity of SOD and levels of sulfhydryl groups were significantly enhanced in HSD rats as well suggesting a compensatory response to oxidative damage.Reference Pisoschi and Pop55 Although the lipid peroxidation measured by MDA levels using the TBARS assay is a flawed marker, it remains widely used and continues to be commonly employed in current research.Reference Conceição, Franco and Oliveira47,Reference Rolnik, Olas and Szablińska-Piernik48 Moreover, MDA levels using the TBARS method was not the only oxidative damage marker we assessed; we also evaluated carbonylated proteins, and antioxidant defenses (SOD, CAT, and sulfhydryl groups), providing a broader context for understanding oxidative stress that goes beyond MDA levels. This hypothesis is particularly compelling in light of our findings of elevated carbonylated proteins, suggesting that the HSD exacerbates the redox imbalance through additional mechanisms.
While peripheral IR has been extensively studied in the skeletal muscle, WAT, and liver, there is limited information regarding its role in iBAT activation.Reference Schnaider and Borges10 Thus, considering that the SL model appears to attenuate IR in rats fed an HSD, we investigated IR at the tissue level to better understand the underlying regulatory mechanisms and insulin signaling pathways in iBAT. Our data indicated that the SL model mitigated HSD-induced IR in iBAT by up-regulating p-AKT (Ser 473).
First, we demonstrated that an HSD induced iBAT IR, decreasing the p-AKT (Ser 473) expression in these rats. Our findings align with existing literature that also reports a reduction in p-AKT (Ser 473) associated with IR and glucose intolerance.Reference Corbi, Conti and Russomanno24 As a result, the decreased sensitivity to insulin in iBAT reduces the oxidation of circulating glucose, leading to persistent hyperglycemia and exacerbated IR.Reference Lettieri-Barbato9
The increase in p-AKT (Ser 473) observed in the SL model fed an HSD suggests that iBAT activates non-shivering thermogenesis and utilizes circulating glucose and FFA as substrates, thereby mitigating systemic IR. This hypothesis is compelling, as improved insulin sensitivity inhibits hormone-sensitive lipase (HSL) and decreases lipolysis,Reference Xiao, Williams and Grayson15 resulting in lower levels of FFA and PKA-mediated changes observed in the iBAT of the HSD-SL group. Zhang et al. 2021, demonstrated that PI3K/AKT signaling in insulin-sensitive adipocytes stimulates phosphodiesterase activity in vitro, which inhibits PKA-dependent activation of β-adrenergic receptors.Reference Zhang, Yang and Xiang56 Conversely, HSL-knockout rats exhibit altered iBAT morphology without impairing thermogenic function, suggesting that iBAT depends on circulating substrates to maintain its activity during fasting.Reference Schreiber, Diwoky and Schoiswohl57 Accordingly, although lipid content was reduced in the iBAT of HSD-SL rats, its activity may have been preserved through alternative regulatory mechanisms involving the availability of circulating glucose and FFAs.Reference Lettieri-Barbato9 This preservation could contribute to the reduction of systemic IR. Notably, despite non-esterified fatty acids being measured in the serum, the FFA content within iBAT was also reduced. This is particularly relevant as all iBAT analyses were performed using samples collected under fed-state conditions. This approach better reflects the tissue’s metabolic activity in response to nutritional stimuli and reduces confounding effects associated with fasting-induced systemic lipolysis.Reference Kersten34
Moreover, we observed an upregulation of UCP1 expression independent of PGC1-α in rats fed an HSD. This finding may be associated with the increased FFA content in iBAT, as FFAs are known to act both as substrates and allosteric activators of UCP1, thereby contributing to the regulation of its activity.Reference Li, Fromme and Klingenspor58 Although β3-adrenergic signaling is known to stimulate PKA-mediated lipolysis and Ucp1 transcription via CREB,Reference Sanchez-Gurmaches, Tang and Jespersen59 we did not directly assess β3-adrenergic receptor activation or intracellular cAMP levels. However, we did evaluate PKA signaling through the detection of phosphorylated serine/threonine substrates, which may indicate upstream pathway activation. Given that CREB expression was not evaluated in this study, conclusions regarding the complete engagement of the β-adrenergic pathway remain speculative. Nonetheless, the increase in UCP1 expression observed under HSD conditions suggests a potential involvement of this pathway; however, further investigations are warranted to elucidate the mechanism underlying UCP1 regulation, which remains beyond the scope of the current study.
In our study, the SL model was associated with a reduction in total PGC1-α expression. However, it paradoxically promoted the upregulation of UCP1 and morphological features consistent with iBAT hyperplasia, even in the presence of reduced tissue mass and lipid content. These findings suggest that early-life metabolic imprinting induced by SL may prime iBAT towards a more metabolically active phenotype under nutritional challenges, such as exposure to HSD.Reference de Queiroz, Evangelista and Guerra-Sá20 This contrasts with previous literature, which associated SL-induced programming with impaired insulin sensitivity, reduced thermogenic capacity, and decreased levels of β3-adrenergic receptor and UCP1 expression in adulthood.Reference Isganaitis, Woo and Ma16,Reference Dąbrowska and Mirabegron49 In our model, SL rats fed a STD exhibited increased phosphorylation of PKA substrates in iBAT, despite unaltered FFA content, along with increased UCP1 and reduced PGC1-α expression. To account for variations in tissue size, UCP1 expression was normalized to total protein; however, these molecular changes may reflect adaptive signaling rather than a definitive enhancement of thermogenic output. Furthermore, while housing conditions were not adjusted to thermoneutrality (∼30 °C for rats), a factor known to influence sympathetic tone and iBAT recruitment,Reference Fischer, Cannon and Nedergaard60 all groups were maintained under identical ambient temperatures (∼22–24 °C), minimizing potential thermal biases across groups.
Taken together, our findings point toward an adaptive reprogramming of iBAT in SL rats, possibly promoting resilience to HSD-induced metabolic stress. While the increase in UCP1 expression supports this hypothesis, the absence of functional readouts such as oxygen consumption or energy expenditure limits definitive conclusions regarding thermogenic activation. Nonetheless, these findings suggest that SL may modulate key regulators of mitochondrial biogenesisReference Cao, Daniel and Robidoux61 and metabolic plasticity, potentially through mechanisms beyond classical β3-adrenergic input. These include alternative cAMP-mediated pathwaysReference London and Stratakis62 and the MKK3–p38 MAPK signaling cascade, which promotes ATF-2 phosphorylation and subsequent transcriptional activation of PGC1-α.Reference Schreiber, Diwoky and Schoiswohl57 This illustrates the complexity of metabolic imprinting and offers a broader perspective on how early-life nutritional experiences can influence long-term iBAT function.
Regarding redox processes, the SL model exhibited increased CAT enzymatic activity, modulated sulfhydryl levels, and reduced lipid oxidative damage, as evidenced by decreased levels of MDA. In addition, literature suggests an inverse correlation between UCP1 expression and ROS production,Reference Ziqubu, Dludla and Mthembu54 highlighting UCP1’s role in regulating ROS levels and protecting against oxidative stress in iBAT.Reference Zhang, Yang and Xiang56,Reference Schreiber, Diwoky and Schoiswohl57 However, the precise mechanisms by which UCP1 exerts its antioxidant effects remain to be fully elucidated. Several hypotheses have been proposed, including the reduction of “reverse uncoupled respiration”, mitochondrial hyperpolarization, and UCP1 activation by oxidative stress products.Reference Shabalina, Vrbacký and Pecinová63 Nevertheless, further research is needed to understand these phenomena.
Conflicting reports exist regarding the association between the SL model, oxidative stress, and the development of diseases in adulthood. For instance, a study by Conceição et al. 2013, observed alterations in antioxidant ratios and oxidative markers in SL rats, revealing a significant increase in ROS levels due to lipid oxidation after eight weeks, although this was not related to protein oxidation.Reference Conceição, Moura and Trevenzoli50 Our findings, which include IR attenuation, iBAT hyperplasia, upregulation of UCP1 protein, and protection against lipid oxidative damage (as indicated by reduced MDA levels using the TBARS analysis), support the protective effects of the SL in rats fed HSD. This model may help prevent HSD-induced iBAT dysfunction by supporting UCP1 expression, which could facilitate the oxidation of circulating FFA and glucose, reduce oxidative damage, and contribute to improved insulin sensitivity.
While our findings are significant in the context of metabolic programming, some limitations should be considered. First, our study only included male rats, which limits the ability to assess potential sex-specific responses to early-life nutritional programming. Given the known differences in energy metabolism and adipose tissue regulation between sexes,Reference Sánchez-Garrido, Castellano and Ruiz-Pino64,Reference Palou, Priego, Sánchez, Palou and Picó65,Reference Costa, Andreazzi and Bolotari66 future studies must include both male and female animals to validate and generalize our findings. Second, although the SL model is widely used to induce early-life overfeeding, it may also impact other developmental aspects, such as thermoregulatory behavior during the suckling period.Reference Teicher and Kenny67,Reference Enes-Marques and Giusti-Paiva68 Pups from SL experience less huddling, which could increase individual thermogenic demand and confound interpretations related to iBAT programming.Reference Teicher and Kenny67,Reference Enes-Marques and Giusti-Paiva68 While our analysis primarily emphasizes the nutritional factors involved in SL-induced metabolic imprinting, the potential contribution of altered thermoregulatory stimuli during lactation cannot be completely excluded. Despite these limitations, our findings provide valuable insights into how early-life nutritional cues can shape long-term metabolic adaptations in iBAT. Importantly, these limitations also guide the design of future investigations that will aim to disentangle the relative contributions of nutrition, thermoregulation, and sex to the metabolic outcomes associated with litter size manipulation.
Conclusion
The present study demonstrated that the SL model induces metabolic imprinting, activating molecular markers associated with iBAT activation, such as increased expression of UCP1 and p-AKT (Ser473), and possibly enhancing FFA oxidation. Additionally, the SL model appears to mitigate oxidative damage caused by an HSD. These findings provide novel insights into the potential role of iBAT in promoting glucose tolerance and metabolic flexibility. While our results challenge previous reports in the literature, they suggest that the long-lasting metabolic imprinting induced by the SL model may enhance the organism’s plasticity to dietary changes across the lifespan. It is important to note, however, that our conclusions rely on indirect molecular evidence, as we did not assess thermogenic function through direct measurements, such as oxygen consumption or heat production. While the observed molecular changes may suggest a possible protective effect of SL against HSD-induced IR in adulthood, further studies are needed to clarify the underlying mechanisms and confirm these findings.
Acknowledgements
The authors thank the Animal Science Center (CCA) at the Federal University of Ouro Preto for providing animals for this study and acknowledge FINEP and FAPEMIG (APQ-02200-21, APQ-02029-21, APQ-04983-22, APQ-02511-22) for their support. Thanks also to the Advanced Microscopy and Microanalysis Multi-User Laboratory, NUPEB, UFOP, and research support foundations CAPES and CNPq. We appreciate Editage (www.editage.com) for English language editing and Raphael Antônio Borges Gomes for his assistance with the experiments.
Financial support
This work was supported by the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG APQ-02200-21). Additionally, this work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, funding code 001).
Competing interests
The authors declare that there are no conflicts of interest.
Ethical standard
All experimental protocols necessary for the development of this work were approved by the Ethics Committee for the Use of Animals at the Federal University of Ouro Preto (UFOP) (protocol number 2245040518).
