Introduction
Programming periods, such as gestation, lactation, and puberty, are markedly known by the phenotypic plasticity, increasing the risk of cardiometabolic diseases late in life. Reference Fernandez-Twinn and Ozanne1 The “Developmental Origins of Health and Disease” (DOHaD) concept predicts that adaptations during critical developmental periods determine health or illness in adulthood. Reference Barker2
Puberty is physiologically characterized by sexual and physical maturation, and an adequate nutritional support is necessary for a normal development. Reference Das, Salam and Thornburg3 Nutritional disorders, such as overnutrition or undernutrition during phenotypic plasticity periods program to obesity, diabetes, and cardiovascular disorders. Reference de Oliveira, de Moura and Miranda4–Reference Chavaglia Cavalet, dos Santos Ribeiro and Rosa6 Epidemiological and experimental studies have demonstrated that a high-fat (HF) diet induces overweight/obesity, hyperphagia, insulin resistance, glucose intolerance, hepatic dysfunctions, and other disturbances. Reference Seong, Kang, Sun and Kim7–Reference Buettner, Schölmerich and Bollheimer10 Specifically, HF intake during the puberty induces abnormalities in reproductive function, adiposity, and impairment of hippocampal-dependent memory associated with enhanced hippocampal inflammation later in life in male rats. Reference Ibáñez, Erthal and Ogo5,Reference Boitard, Cavaroc and Sauvant11 Moreover, prepubertal HF female rats had polycystic ovary syndrome. Reference Patel and Shah12 In humans, eating behavior among adolescents contribute to obesity onset and related disorders. Reference Moreno, Rodríguez and Fleta13,Reference Simmonds, Burch and Llewellyn14
Eating habits and obesity are important risk factors for liver diseases, such as nonalcoholic fatty liver disease (NAFLD), characterized by fat accumulation in the liver accompanied by different degrees of inflammation and fibrosis. Reference Wesolowski, Kasmi, Jonscher and Friedman15 Among adolescents, the prevalence of NAFLD is high and increases the risk of glucose dyshomeostasis. Reference Welsh, Karpen and Vos16,Reference Armstrong, Adams, Canbay and Syn17 In addition, NAFLD is more common in men than in women. Reference Tsuneto, Hida and Sera18 Several molecular mechanisms have been proposed to explain the development of NAFLD, although it is not fully understood. Studies suggest that the capacity of the liver is overwhelmed by excess substrates, leading to the accumulation of lipotoxic lipids and consequent hepatocellular injury. Reference Friedman, Neuschwander-Tetri, Rinella and Sanyal19 Liver lipotoxicity may be related to failure in gluconeogenesis and/or lipogenesis processes, impacting hepatic lipid uptake, synthesis, oxidation, and export of fat. HFD fed animals increased the expression of PEPCK, that contributes with high blood glucose in this animal model. Reference Arçari, Santos, Gambero, Ferraz and Ribeiro20,Reference Kim, Quan and Jeong21 On the other hand, in mouse livers lacking cytosolic phosphoenolpyruvate carboxykinase (PEPCK), an enzyme responsible for converting cytoplasmic oxaloacetate into phosphoenolpyruvate (PEP), intermediates of the tricarboxylic acid (TCA) cycle accumulate, and hepatic steatosis is observed. Reference Burgess, Hausler and Merritt22 Increased hepatic de novo fatty acid synthesis and upregulation of key lipogenic enzymes, such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), are also observed in clinical and experimental models of NAFLD. Reference Geisler and Renquist23 Maternal HF diet induces molecular alterations in pathways of lipid metabolism in the liver in offspring rats during the puberty. Reference Oliveira, Souza and Souza24 When these same animal models are exposed to high-fructose diet during puberty, liver injury is intensified, demonstrating that this period of life is extremely susceptible to the metabolic programming. Reference Oliveira, Caetano and Miranda25 Regarding exposure to HF, some aspects of its effects remain unclear, especially in relation to puberty as a window of programming. We hypothesized that the exposure to HF during puberty can induce liver dysfunction at adulthood, promoting fat accumulation. Thus, we investigated the long-term repercussions of HF during puberty on (1) body weight, food intake and adiposity, (2) hepatic morphology and functions, (3) glucose homeostasis, and (4) protein content of PEPCK, ACC, and FAS. In addition, insulin sensitivity in adult rats were also investigated.
Materials and methods
Ethical approval
The handling of animals and experimental procedures were performed according to the rules of the National Council of Animal Experiments Control (CONCEA) and the Brazilian Society of Science in Laboratory Animals (SBCAL) and approved by the Ethics Committee on Animal Use of State University of Maringá - CEUA/UEM (protocol number 1527130815).
Experimental design and diets
Twenty-five-day-old male Wistar rats were housed in the Animal Facility of the Laboratory of Secretion Cell Biology, Department of Biotechnology, Genetic and Cell Biology of State University of Maringa, in polypropylene cages (45 × 30 × 15 cm) maintained on a 12 h light–dark cycle (07:00 lights on) and controlled temperature (22.0 ± 2°C). At the postnatal day 30 (PND30), after 5 days of environmental adaptation, the animals were divided into two groups: control group fed a standard chow, normal fat diet (NF; 15.66 kJ/g; n = 40), and rats fed a HF (HF; 22.46 kJ/g; n = 40). Table 1 shows the composition of both diets. Diets were manufactured as previously described Reference Gomes, Tófolo and Rinaldi26 and were given ad libitum during 30 days, from PND30 until PND60. Subsequently, from PND60 to PND120 both NF and HF groups were fed with standard chow. Fig. 1a summarizes the experimental protocol. Animals had unlimited access to food and water. The animals were maintained in cages with 3 to 4 animals. Body weight and food intake were evaluated weekly throughout the experimental period.
Figure 1. Long-term effects of high-fat diet during puberty on food intake and body composition of adult rats. Experimental protocol (A), curve of body weight throughout experimental period (B), representative images showing the obese phenotype of the HF rat (C), food intake throughout experimental period (D), area under curve of food intake from PND30 until PND60 (E), area under curve of food intake from PND60 until PND120 (F), energy intake throughout experimental period (G), area under curve of energy intake from PND30 until PND60 (H), area under curve of energy intake from PND60 until PND120 (I), periepididymal fat (J), retroperitoneal fat (K), and inguinal fat (L) at PN120. Data are presented as the mean ± SEM. Two-way ANOVA (B, D, and G) *p < 0.05; Student’s t-test (bar graphs) *p < 0.05, **p < 0.01, and ***p < 0.001.
Table 1. Composition of diets
Intravenous glucose tolerance test
At PND120, a cohort of adult offspring (n = 10 per group) from both experimental groups were anesthetized (ketamine 75 mg + xylazine 15 mg/kg of BW, i.m.) and a silicone cannula (Silastic, Dow Corning, Midland, MI, USA) was implanted in the right jugular vein. After overnight fasting, an intravenous glucose tolerance test (ivGTT) was performed in conscious rats, as previously described. Reference Gomes, Bueno and Schamber27 Blood samples were collected and centrifuged at 10,000 r.p.m. for 5 min for plasma collection, and then the plasma was stored at −20 °C for subsequent measurements of glucose and insulin. Animals used for the ivGTT were not used in any other experimental procedures.
Intraperitoneal insulin tolerance test
At PND120, another cohort of adult offspring (n = 10 / group) from both experimental groups received an intraperitoneal injection of human recombinant insulin (2 U/kg of BW, Eli Lilly, São Paulo, Brazil). The Intraperitoneal insulin tolerance test (ipITT) was performed in fed and conscious rats. Blood samples were collected through a small cut at the tip of the tail, and blood glucose was measured using a glucometer (ACCU-CHEK Advantage, Roche Diagnostics, Mannheim, Germany). Blood samples were collected at 0 (prior to insulin injection) and 5, 10, 15, 20, and 25 min after injection, and glycemia was measured at each corresponding time. Thereafter, the constant rate for glucose decay (Kitt) was calculated by the formula Kitt (%/min) = 0.693/t1/2. The plasma glucose t1/2 was calculated from the slope of the least-squares analyses of plasma glucose concentrations during the linear decay phase. Reference Miranda, da Silva Franco and Previate28 Animals used in this test were not used in any other experimental procedures.
Intraperitoneal pyruvate tolerance test
Another cohort of adult offspring (PND120, n = 10 / group) was fasted for 16 hours and submitted to an intraperitoneal pyruvate tolerance test (ipPTT). Conscious rats received an intraperitoneal injection of 25% (w/v) sodium pyruvate solution diluted in normal saline (2 g / kg of BW; Sigma-Aldrich, St Louis, MO, USA). Blood samples were collected at 0 min (before pyruvate injection) and 15, 30, 60, 90, and 120 min after injection. Blood samples were collected through a small tail tip cut, and blood glucose was measured using a blood glucose meter (ACCU-CHEK Advantage, Roche Diagnostics, Mannheim, Germany). Reference Milanski, Arruda and Coope29 Animals used for the ipPTT were not used in any other experimental procedures.
Euthanasia and sample collection
At PND120, another cohort of fasted animals (n = 10 / group) was anesthetized with sodium thiopental (40 mg/kg of BW, i.p.; Thiopentax, Cristália, Brazil) and euthanized by exsanguination for blood, liver, and white adipose tissue (WAT) sample collection.
Liver morphological analyses
Liver samples were removed, frozen in liquid nitrogen, and stored in a -80 °C freezer. Subsequently, these samples were embedded in Tissue-Tek (Sakura Finetek, Torrance, CA, USA). Nonserial histological sections (10 µm thick) were obtained using a Leica CM1850 cryostat (Leica Biosystems, Wetzlar, Germany) and stained with Oil Red O. The percentage of lipid inclusion was determined by the ratio between the red oil marked area and total area of the images (% = marked area / total area × 100). Morphological analyses were performed using digital images (TIFF 24-bit color, 2560 × 1920 pixels) obtained with a light microscope (Olympus BX41, Tokyo, Japan) and a camera QColor 3 Olympus (×400 magnification). The images were acquired randomly from 5 rats per group (20 images per rat). The analyses were performed using the software ICY (Institut Pasteur, Paris, France).
Immunohistochemistry
Pancreas samples were fixed in 10% buffered formalin, dehydrated, embedded in histological paraffin, and sectioned (5 μm) in nonserial cuts. Sections were incubated for 60 min at room temperature with a specific mouse monoclonal primary antibody diluted to 1:500 (anti-insulin - catalogue # I2018 from Sigma-Aldrich Inc., St Louis, MO, USA). The immunohistochemistry assay was performed using a commercial kit (Histostain-Plus, Invitrogen, Carlsbad, CA, USA), as previously described. Reference Gomes, Bueno and Schamber27 Finally, sections were counterstained with hematoxylin. The morphometric analyses were performed using digital images (TIFF 24-bit color, 2560 × 1920 pixels). Quantitative analyses of the pancreatic islets number were performed using digital images (×20 magnification) from 6 different fields from each animal. Analyses of the pancreatic islet area and pancreatic β-cell mass were performed using 40 digital images (×400 magnification) from each animal. Analyses were performed using Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, MD, USA).
Biochemical and hormonal analyses
Blood samples were centrifuged (10,000 r.p.m. for 5 min), and plasma used to determinate blood glucose, triglycerides, and total and HDL cholesterol by the enzymatic-colorimetric method with a commercial kit (Gold Analisa, Belo Horizonte, Minas Gerais, Brazil) according to the manufacturer’s instructions. Blood insulin was measured by radioimmunoassay in a gamma counter (Wizard2 Automatic Gamma Counter, TM-2470, PerkinElmer, Shelton, CT, USA). It was used as standards human insulin and anti-insulin antibody (catalogue # I9278 and 4200691 Sigma-Aldrich, St Louis, MO, USA) and recombinant human insulin 125I-labeled (catalogue # NEX420 PerkinElmer, Shelton, CT, USA). The intra-assay coefficients of variation were in the range of 8–10%. The limit of detection was 0.006 ng/ml. The measurements were taken in a single assay.
Western blotting
Liver samples (n = 4 / group) were homogenized in lysis buffer (PBS [137 mM NaCl, 2.4 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4], 8.8 mM IGEPAL CA-630, 12 mM sodium deoxycholate, 3.47 mM SDS, 2 mM Na3VO4, 1 mM PMSF, 2.34 μM leupeptin, 0.154 μM aprotinin, 1.45 μM pepstatin) in a glass homogenizer at 4°C and centrifuged at 10,000 r.p.m. at 4°C for 20 minutes. After, total protein content was quantified by the bicinchoninic acid method (Sigma-Aldrich, Missouri, EUA) according to the manufacturer’s instructions. Samples were denatured in Laemmli buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 1% 2-mercaptoethanol, 0.001% bromophenol blue). Aliquots of 30 μg of protein from each sample were subjected to separation by SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Amersham Protran, GE Healthcare, Little Chalfont, BUX, UK), The membranes were incubated with a blocking solution (5% skim powdered milk, 10 mM Tris, 150 mM NaCl, 0.02% Tween 20) under mild agitation for 90 min at room temperature. Subsequently, the membranes were washed with TBS and then incubated overnight at 4°C with the following specific primary antibodies: PEPCK (at 1:1000 dilutions; catalogue # sc-32879 from Santa Cruz Biotechnology, CA, USA), ACC (at 1:1000 dilutions; catalogue # 4190 from Cell Signalling Technology MA, USA), FAS (at 1:1000 dilutions; catalogue # 3180 from Cell Signalling Technology MA, USA), β-actin (at 1:1000 dilution; catalogue # A2228 from Sigma-Aldrich MO, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH at 1:1000 dilution; catalog # SC-25778 from Santa Cruz Biotechnology, CA, USA). Then, the membranes were gently washed (3x 5 min; 10 mM Tris, 150 mM NaCl, 0.02% Tween 20) and incubated with the manufacturer-specific HRP-conjugated secondary antibody (anti-rabbit at 1:10,000 dilution; catalogue # B7389 from Sigma-Aldrich MO, USA) or (anti-mouse at 1:10,000 dilution; catalogue # B8520 from Sigma-Aldrich MO, USA) for 90 min. The protein bands were visualized with chemiluminescence (ECL kit; Amersham Biosciences, London, UK) and exposed to ImageQuant LAS (GE Healthcare, Buckinghamshire, UK). The area and density of the bands were quantified by ImageJ software (NIH, MA, USA). β-Actin and GAPDH were used as a load control.
Statistical analysis
Data are expressed as Mean ± standard error of the mean (SEM). To compare differences between groups unpaired Student’s t-test was used. To compare the differences in body weight, food intake, ivGTT, ipITT, and ipPTT curves, two-way ANOVA was used followed by the Sidak post hoc test. GraphPad Prism 9 software (v9.01, GraphPad, San Diego, CA, USA) was used for data analyses and build graphics. Differences were considered significant at p < 0.05.
Results
Long-term effects of HF diet during puberty on food intake and body composition
As shown in Fig. 1b, during HF intake period (PND30 – PND60) animals from both groups had no change in the body weight. However, at PND70 we observed a clear increase on the body weight of HF animals compared to NF animals (NF 334.70 ± 5.39 g vs HF 360.70 ± 5.60 g; p < 0.05), which gradually increased throughout experimental period, with a difference lasting up to PN120 (NF 413.10 ± 12.30 g vs HF 459.60 ± 10.00 g; p < 0.05).
As depicted in Fig. 1d–f, during the treatment (PND30 – PND60), HF animals had low food intake compared to NF animals (AUC food intake NF 224.90 ± 4.22 g vs HF 146.90 ± 12,89 g; p < 0.0001). However, from PND60 until PND120, HF animals had higher food intake (AUC food intake NF 94.68 ± 13.16 g vs HF 408.00 ± 59.79 g n = 4; p < 0.05).
In the same sense, the energy intake of HF animals was similar than that of their counterparts during the dietary intervention (AUC of energy intake 30–60 days NF 543.2 ± 100.1 vs HF 669.3 ± 86.5; Fig. 1h), however after the dietary intervention the energy intake of HF group was lower than NF group (AUC of energy intake 60-120 days NF 1562.0 ± 96.7 vs HF 586.1 ± 108.4; p < 0.001; Fig. 1i).
For WAT mass, we observed an effect of programming as shown in representative Fig. 1c, and it was confirmed by quantitative measurements. HF animals had higher periepididymal fat (NF 5.52 ± 0.41 g vs HF 7.01 ± 0.47 g; p < 0.05; Fig. 1j), retroperitoneal fat (NF 5.56 ± 0.59 g vs HF 7.76 ± 0.55 g; p < 0.01; Fig. 1k), and inguinal fat stores (NF 1.71 ± 0.28 g vs HF 2.99 ± 0.52 g; p < 0.05; Fig. 1l).
Long-term effects of HF diet during puberty on glucose homeostasis and pancreatic islets morphology
HF animals showed increased (+13%) fasting blood glucose (NF 71.78 ± 4.05 mg/dL vs HF 80.70 ± 1.50 mg/dL; p < 0.05; Fig. 2a) and had higher (+69%) fasting blood insulin (NF 0.48 ± 0.06 ng/mL vs HF 0.81 ± 0.12 ng/mL; p < 0.05; Fig. 2d) compared to NF animals.
Figure 2. Long-term effects of high-fat diet during puberty on glucose homeostasis of adult rats. Fasting blood glucose (A), curve of blood glucose during ivGTT (B), AUC of blood glucose (C), fasting blood insulin (D), curve of blood glucose during ipITT (E), Kitt (F), pancreas mass (G), representative photomicrographs (×100 magnification, scale bars = 200 μm) shows pancreas sections immunostained with anti-insulin antibody (H), quantitative analyses of the islet number (I), islet area (J), and β-cell mass (K). Data are presented as the mean ± SEM. Two-way ANOVA (B and E) *p < 0.05; Student’s t-test (bar graphs) *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
As depicted in Fig. 2b, HF animals had higher blood glucose levels during ivGTT compared to NF animals, leading to increased (+56%) area under the curve of the test (NF 1408 ± 196.8 vs HF 2200 ± 283.2 p < 0.05; Fig. 2c). In addition, HF animals had higher blood glucose levels during ipITT (Fig. 2e). On the other hand, Kitt was significantly lower (−34%) in HF animals than in your counterparts (NF 2.26 ± 0.11 %/min vs HF 1.49 ± 0.28 %/min; p < 0.05; Fig. 2f).
The pancreas mass of the HF animals was heavier than the pancreas mass of NF group (Fig. 2g; NF 0.76 ± 0.02 g vs HF 0.99 ± 0.09 g; p < 0.05). Also, HF animals showed high number (NF 2.68 ± 0.12 islets/mm2 vs HF 4.34 ± 0.16 islets/mm2; Fig. 2i; p < 0.0001) and hypertrophied pancreatic islets (NF 85.80 ± 12.61 x103 μm2 vs HF 177.4 ± 13.02 x103 μm2; Fig. 2j; p < 0.001), with high beta cell mass (NF 1.41 ± 0.08 % vs HF 2.21 ± 0.12 %; Fig. 2k; p < 0.0001).
Long-term effects of HF diet during puberty on gluconeogenesis, lipid profile, liver morphology, and key-enzymes expression in the liver of adult rats
As depicted in Fig. 3a, HF animals had higher (+18%) liver mass compared to NF animals (NF 14.26 ± 0.48 g vs HF 16.85 ± 0.44 g; p < 0.01). Moreover, HF animals showed increased blood glucose during ipPTT (Fig. 3b), leading to higher (+55%) AUC compared to NF animals (NF 3247 ± 390.20 vs HF 5040 ± 534.00; p < 0.05; Fig. 3c), suggesting an increase in the hepatic gluconeogenesis.
Figure 3. Long-term effects of high-fat diet during puberty on gluconeogenesis, lipid profile, liver morphology, and key-enzymes expression in the liver of adult rats. Liver mass (A), curve of blood glucose during ipPTT (B), AUC of blood glucose (C), blood triglycerides levels (D), total cholesterol (E), HDL cholesterol (F), steatosis area in the liver (G), representative photomicrographs (×400 magnification, scale bars = 50 µm) showing steatosis area in the liver stained with red oil (H). Western blot analysis of PEPCK (I), ACC (J), and FAS (K) and in the liver of the NF and HF rats. Representative immunoblots of proteins are show above the graphs. β-actin and GAPDH were used as a loading control. Data are presented as the mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (B) *p < 0.05; Student’s t-test (bar graphs) *p < 0.05, **p < 0.01, and ****p < 0.0001.
HF animals also presented higher (+34%) blood triglycerides (NF 155.90 ± 6.74 mg/dL vs HF 209.60 ± 19.41 mg/dL; p < 0.05; Fig. 3d), with no changes in total (NF 90.22 ± 4.17 mg/dL vs HF 95.00 ± 6.15 mg/dL; p < 0.05; Fig. 3e) or HDL cholesterol (NF 52.78 ± 3.04 mg/dL vs HF 56.00 ± 2.94 mg/dL; p < 0.05; Fig. 3f) when compared to NF group. Besides, HF animals also showed an increase of 10-fold in lipid inclusion in the liver compared with the NF animals (NF 1.13 ± 0.23 % vs HF 11.18 ± 0.81 %; p < 0.01; Fig. 3g–h).
HF animals also showed 79% higher protein content of PEPCK (NF 100.00 ± 10.47 % of control vs HF 178.70 ± 46.07 % of control; p < 0.05; Fig. 3i). Regarding hepatic biomarkers of lipogenesis at PND120, HF animals had no change in protein content of ACC compared to NF animals (NF 100.00 ± 18.81 % of control vs HF 107.30 ± 13.24 % of control; p = 0.761; Fig. 3j). However, HF animals showed 37% higher protein content of FAS compared to NF animals (NF 100.00 ± 10.43 % of control vs HF 137.20 ± 8.85 % of control; p < 0.05; Fig. 3k).
Discussion
Traditionally, there is a strong emphasis on the role of the fetal period in metabolic disease programming; however, there is a growing interest in understanding other programming windows. As already mentioned, puberty is also a period of intense neuroendocrine and reproductive changes Reference Ebling30,Reference Salam, Bhutta, Koletzko, Bhatia and Bhutta31 and could be linked to disorders later in life. Studies have shown that eating patterns during adolescence have changed in the last two to three decades and have affected body weight gain, which can contribute to overweight and obesity from this period of life. Reference Moreno, Rodríguez and Fleta13 In the current study, we demonstrated that HF consumption during puberty can induce metabolic dysfunctions in rats at adulthood. HF animals showed obese phenotype, hyperphagia, hypertriglyceridemia, higher liver mass, hepatic steatosis, hepatic insulin resistance and PEPCK and FAS overexpression. Fig. 4 summarizes the main findings of our study and the proposed mechanism for the observed changes. To our knowledge, our results are the first to demonstrate metabolic programming of hepatic dysfunction in this experimental model induced by HF during puberty.
Figure 4. Summary of key findings about hepatic metabolic long-term programming in adult male Wistar rats in response to HF intake during puberty. High-fat diet during puberty led to an increase in white adipose tissue. Increased WAT led to increased circulating free fatty acids, and insulin resistance. Peripheral insulin resistance favored increased insulinemia and hypertrophy of pancreatic β-cells. In the liver, insulin resistance led to greater activation of the gluconeogenic pathway, which provides substrates for lipogenic pathway activity (ACC/FAS). Lipogenesis, combined with a higher amount of free fatty acids and insulin resistance, led to hepatic steatosis. ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; FFA, free-fat acids; HFD, high-fat diet; NAFLD, nonalcoholic fatty liver disease; PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; TCA, tricarboxylic acid; WAT, white adipose tissue.
It is also important to highlight that the decreased food intake observed during HF challenge (PND30-60) is associated with the high caloric content of this diet and the lower palatability, which induces satiety faster than a standard diet. In fact, we prove that HF-fed animals ingested lower amounts of energy at the end of dietary intervention. However, the energy intake after dietary intervention was similar, when corrected to body weight, in both groups. In addition, the hyperphagia observed in HF group later in life could be an effect of hypothalamic inflammation, as previously observed in other animal models and in humans, that appears prior to the weight gain, Reference Morselli, Criollo, Rodriguez-Navas and Clegg32–Reference del Fernández-Arjona, León-Rodríguez, Grondona and López-Ávalos34 demonstrating the presence of a programming effect. However, it is a limitation of this study do not assess hypothalamic inflammation, that should be assessed by other studies. Reinforcing that, in a previous study, we showed that exposure to HF during puberty induces long-term obesity. Reference Ibáñez, Erthal and Ogo5 Other studies have also shown these effects in similar HF models. Reference Ullah, Su and Shen35,Reference Holtrup, Church and Berry36
We also observed hyperglycaemia, hyperinsulinemia, and insulin resistance, as suggested by the Kitt results. Other study showed that maternal HF intake during gestational and lactational periods induces hyperinsulinemia and hepatic insulin resistance in offspring. Reference Ashino, Saito and Souza37 Our group also showed hyperinsulinemia and insulin resistance in HF-fed male Wistar rats. Reference Gomes, Tófolo and Rinaldi26 Although those studies differ from our experimental model, it has been well established by the literature that an HF is able to dysregulate glucose and insulin metabolism and could induce the development of diabetes mellitus. Reference Holemans, Caluwaerts, Poston and Van Assche38,Reference Moreno-Fernández, Garcés-Rimón and Vera39 In this sense, we also showed that HF animals presented increased and hypertrophied islets, and higher β-cell mass, that is well described as a signal of systemic insulin resistance.
We hypothesized that rats fed a HF during puberty develop obesity, insulin resistance, and that liver metabolism could be affected. Here, we evaluated hepatic glucose production by intraperitoneal pyruvate administration and, as expected, HF animals had a significant increase in blood glucose levels after pyruvate injection. These increased of glucose levels indicates an increase in hepatic gluconeogenesis and insulin resistance. Corroborating this, PEPCK, an enzyme responsible for converting cytoplasmic oxaloacetate into PEP during gluconeogenesis, was overexpressed in the liver form HF animals. Thus, these results suggest that these animals develop hepatic insulin resistance as well as lower peripheral insulin sensitivity. Some studies in different models have demonstrated that HF alters gluconeogenesis in the liver. Reference Ashino, Saito and Souza37,Reference Maeda Júnior, Constantin and Utsunomiya40 As a limitation of our study, we did not perform any analyses in other peripheral tissues; however, we suggest that HF animals may develop peripheral insulin resistance, reducing glucose uptake by muscle and adipose tissue.
HF animals had higher triglycerides levels and presented increased lipid inclusion in the liver, which characterizes hepatic steatosis. This condition seems to be related to hepatic insulin resistance, which could be suggested by FAS overexpression. The enzymes ACC and FAS are involved in the steps of de novo lipogenesis, and both activities are insulin-regulated. FAS overexpression is known in livers from HF animals. Reference Dorn, Riener and Kirovski41 As ACC and FAS levels were highly sensitive to nutritional and hormonal changes, we hypothesized that disruptions in the plasticity of these enzyme mechanisms observed in this work, confirming the puberty as a programming window. It is possible that ACC expression during HF intake was affected; however, in the long-term, this change disappeared, which did not occur to FAS, demonstrating a plastic effect of the expression of this enzyme, i.e., in the adult HF group. Unfortunately, we did not perform any analyses immediately after HF challenge.
In fact, fat accumulation in the liver is highly associated with insulin resistance and NAFLD progression. Free fatty acids (FFAs) from WAT as well as from de novo lipogenesis and from the diet are responsible for the increase of ectopic fat in the liver. Moreover, hepatic steatosis is also associated with liver inflammation caused by increased FFA. Furthermore, β-oxidation which occurs in liver mitochondria, may also be impaired by the increased FFA load in NAFLD. Reference Sanyal, Campbell-Sargent and Mirshahi42 Some of these interactions were demonstrated in mice fed an HF and indicate that lipid accumulation in the liver leads to subacute hepatic inflammation, causing insulin resistance in the liver and also in other insulin-dependent tissues. Reference Cai, Yuan and Frantz43 Considering that obesity is a public health problem and is a potential condition for the development of NAFLD, Reference Milić, Lulić and Štimac44,Reference Browning, Szczepaniak and Dobbins45 our findings in this study provide evidence of how HF intake during puberty affects metabolic biomarkers and morphology in the long-term, even after the HF intake was interrupted.
Conclusion
In summary, our results demonstrated that HF consumption during puberty increased the susceptibility to obesity and impaired glucose metabolism and liver injury later in life. The exact mechanisms that lead to these dysfunctions still need further investigation. However, hepatic insulin resistance and lipid accumulation directly affect some important proteins involved in gluconeogenesis (PEPCK) and lipogenesis (FAS) and seem to be important contributing factors. Furthermore, considering future clinical investigations, we must consider the poor eating habits of adolescents as an important risk factor for developing obesity and NAFLD later in life.
Acknowledgments
All authors are grateful to Maroly Valentim Alves Pinto for technical assistance.
Financial support
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES) – Finance Code 001.
Competing interests
None.
