Shifting to a control diet after a high-fat, high-sucrose diet intake induces epigenetic changes in retroperitoneal adipocytes of Wistar rats

The aim of the study was to analyze the phenotypic and epigenetic changes induced by the shift to a chow diet after an obesogenic environment. Animals were randomized to fed chow (control group) or high-fat–sucrose diet (HFS). After 10 weeks, half of the rats fed with HFS diet were reassigned to a chow diet (rest group) while the other half continued with the obesogenic diet (HFS group) until week 20. Changes in fat content, biochemical profile, and DNA methylation levels of several gene promoters from retroperitoneal adipocytes were analyzed. HFS diet intake for 10 weeks induced obese phenotype in the animals, increasing body weight and fat content. These effects were maintained until the end of the trial in HFS group, where an increase in liver fat content, a modification of lipid profile, and retroperitoneal adipose tissue hypertrophy were also observed. Changing the dietary pattern reversed these parameters. Epigenetic analysis showed that HFS diet intake for 20 weeks hypermethylated several CpG sites (6.7 and 29.30) and hypomethylated CpG site 15 from leptin gene promoter. Moreover, the obesogenic diet also hypomethylated CpG site 1 from Fasn (fatty acid synthase) gene promoter, without changes on Ppargc1a (peroxisome proliferator-activated receptor gamma coactivator 1-alpha), Srebf1 (sterol regulatory element-binding transcription factor 1), and aquaporin 7. Shifting to a chow diet reverted HFS-induced DNA methylation levels of some CpG sites of leptin promoter. Changing the dietary pattern hypomethylated a CpG site of Srebf1 and hypermethylated other CpGs on Ppargc1a and Fasn promoter. This study shed light on the reversibility of phenotypical and epigenetic changes induced by a HFS diet intake (AU)

Shifting to a control diet after a high-fat, high-sucrose diet intake induces epigenetic changes in retroperitoneal adipocytes of Wistar rats.

The aim of the study was to analyze the phenotypic and epigenetic changes induced by the shift to a chow diet after an obesogenic environment. Animals were randomized to fed chow (control group) or high-fat-sucrose diet (HFS). After 10 weeks, half of the rats fed with HFS diet were reassigned to a chow diet (rest group) while the other half continued with the obesogenic diet (HFS group) until week 20. Changes in fat content, biochemical profile, and DNA methylation levels of several gene promoters from retroperitoneal adipocytes were analyzed. HFS diet intake for 10 weeks induced obese phenotype in the animals, increasing body weight and fat content. These effects were maintained until the end of the trial in HFS group, where an increase in liver fat content, a modification of lipid profile, and retroperitoneal adipose tissue hypertrophy were also observed. Changing the dietary pattern reversed these parameters. Epigenetic analysis showed that HFS diet intake for 20 weeks hypermethylated several CpG sites (6.7 and 29.30) and hypomethylated CpG site 15 from leptin gene promoter. Moreover, the obesogenic diet also hypomethylated CpG site 1 from Fasn (fatty acid synthase) gene promoter, without changes on Ppargc1a (peroxisome proliferator-activated receptor gamma coactivator 1-alpha), Srebf1 (sterol regulatory element-binding transcription factor 1), and aquaporin 7. Shifting to a chow diet reverted HFS-induced DNA methylation levels of some CpG sites of leptin promoter. Changing the dietary pattern hypomethylated a CpG site of Srebf1 and hypermethylated other CpGs on Ppargc1a and Fasn promoter. This study shed light on the reversibility of phenotypical and epigenetic changes induced by a HFS diet intake.

Prenatal stress increases the obesogenic effects of a high-fat-sucrose diet in adult rats in a sex-specific manner.

Stress during pregnancy can induce metabolic disorders in adult offspring. To analyze the possible differential response to a high-fat-sucrose (HFS) diet in offspring affected by prenatal stress (PNS) or not, pregnant Wistar rats (n = 11) were exposed to a chronic mild stress during the third week of gestation. The aim of this study was to model a chronic depressive-like state that develops over time in response to exposure of rats to a series of mild and unpredictable stressors. Control dams (n = 11) remained undisturbed. Adult offspring were fed chow or HFS diet (20% protein, 35% carbohydrate, 45% fat) for 10 weeks. Changes in adiposity, biochemical profile, and retroperitoneal adipose tissue gene expression by real-time polymerase chain reaction were analyzed. An interaction was observed between HFS and PNS concerning visceral adiposity, with higher fat mass in HFS-fed stressed rats, statistically significant only in females. HFS modified lipid profile and increased insulin resistance biomarkers, while PNS reduced insulin concentrations and the homeostasis model assessment index. HFS diet increased gene (mRNA) expression for leptin and apelin and decreased cyclin-dependent kinase inhibitor 1A and fatty acid synthase (Fasn), whereas PNS increased Fasn and stearoyl-CoA desaturase1. An interaction between diet and PNS was observed for adiponutrin (Adpn) and peroxisome proliferator-activated receptor-γ coactivator1-α (Ppargc1a) gene expression: Adpn was increased by the PNS only in HFS-fed rats, whereas Ppargc1a was increased by the PNS only in chow-fed rats. From these results, it can be concluded that experience of maternal stress during intrauterine development can enhance predisposition to obesity induced by a HFS diet intake.

Transcriptomic and epigenetic changes in the hypothalamus are involved in an increased susceptibility to a high-fat-sucrose diet in prenatally stressed female rats.

Disturbances in the prenatal period are linked to metabolic disorders in adulthood, implying the hypothalamic systems of appetite and energy balance regulation. In order to analyze the central effects of a high-fat-sucrose (HFS) diet in prenatally stressed (PNS) female adult rats, Wistar dams were exposed to chronic-mild-stress during the third week of gestation and were then compared with unstressed controls. Adult female offspring were fed a chow or HFS diet for 10 weeks. Changes in body weight, adiposity as well as expression and methylation levels of selected hypothalamic genes were analyzed. PNS induced lower birthweight and body length with no changes in body fat mass. After the HFS diet, the expected overweight model was observed accompanied by higher adiposity and insulin resistance, which was worsened by PNS. The stress model induced higher energy intake in adulthood. Hypothalamic gene expression analysis revealed that the HFS diet decreased Slc6a3 (dopamine active transporter), NPY (neuropeptide Y) and IR (insulin receptor) and increased POMC (pro-opiomelanocortin). Hypothalamic DNA methylation levels in the promoter region of Slc6a3 revealed that Slc6a3 was hypermethylated by the HFS diet in CpG site -53 bp to the transcription start site. HFS diet also hypermethylated CpG site -167 bp of the POMC promoter only in nonstressed animals. No correlations were found between gene expression and DNA methylation levels. These results imply that early-life stress in females increased predisposition to diet-induced obesity in adulthood.

Regulation by chronic-mild stress of glucocorticoids, monocyte chemoattractant protein-1 and adiposity in rats fed on a high-fat diet.

Stress has been reported as a widespread problem and several studies have linked obesity and inflammation-related diseases. Moreover, the combination of suffering from chronic stress and high energy intake might be related to the onset of some metabolic diseases. To study the possible relationships between stress, inflammatory status and obesity, a chronic-mild stress (CMS) paradigm with a high-fat dietary intake model (Cafeteria diet) was implemented on male Wistar rats for 11 weeks. Stress and dietary intake effects on animal adiposity, serum biochemical as well as glucocorticoids and inflammation markers were all analyzed. As expected, consuming a high-fat diet increased body weight, adiposity and insulin resistance in non-stressed animals. A decrease of total white adipose tissue (WAT) and an increase of fecal glucocorticoids, as well as angiotensinogen, and monocyte chemoattractant protein-1 (MCP-1) expression level in retroperitoneal WAT were found only on control-stressed rats. Regarding the serum MCP-1, a decrease was observed on animals under CMS while being fed Cafeteria diet. Furthermore, 11ß-hydroxysteroid dehydrogenase activity, a glucocorticoid and obesity biomarker in the liver, was influenced by high-fat diet intake but not by stress. Finally, statistical analysis showed a strong relation between MCP-1 expression levels in retroperitoneal WAT, fecal corticosterone and total WAT. This trial proved that CMS induced a glucocorticoid-mediated response, which was reduced by the intake of a Cafeteria diet. These findings suggest that a high-fat diet could protect against a stress condition and revealed a different behavior to a stressful environment depending on the nutritional status.

Influence of dietary macronutrient composition on adiposity and cellularity of different fat depots in Wistar rats

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The aim of this study was to investigate the role of dietary macronutrient content on adiposity parameters and adipocyte hypertrophy/hyperplasia in subcutaneous and visceral fat depots from Wistar rats using combined histological and computational approaches. For this purpose, male Wistar rats were distributed into 4 groups and were assigned to different nutritional interventions: Control group (chow diet); high-fat group, HF (60% E from fat); high-fat-sucrose group, HFS (45% E from fat and 17% from sucrose); and high-sucrose group, HS (42% E from sucrose). At day 35, rats were sacrificed, blood was collected, tissues were weighed and fragments of different fat depots were kept for histological analyses with the new softwareAdiposoft. Rats fed with HF, HFS and HS diets increased significantly body weight and total body fat against Control rats, being metabolic impairments more pronounced on HS rats than in the other groups. Cellularity analyses usingAdiposoft revealed that retroperitoneal adipose tissue is histologically different than mesenteric and subcutaneous ones, in relation to bigger adipocytes. The subcutaneous fat pad was the most sensitive to the diet, presenting adipocyte hypertrophy induced by HF diet and adipocyte hyperplasia induced by HS diet. The mesenteric fat pad had a similar but attenuated response in comparison to the subcutaneous adipose tissue, while retroperitoneal fat pad only presented adipocyte hyperplasia induced by the HS diet intake after 35 days of intervention. These findings provide new insights into the role of macronutrients in the development of hyperplastic obesity, which is characterized by the severity of the clinical features. Finally, a new tool for analyzing histological adipose samples is presented (AU)

High fat diet-induced obesity modifies the methylation pattern of leptin promoter in rats.

Leptin is an adipokine involved in body weight and food intake regulation whose promoter region presents CpG islands that could be subject to dynamic methylation. This methylation process could be affected by environmental (e.g. diet) or endogenous (e.g., adipocyte differentiation, inflammation, hypoxia) factors, and could influence adipocyte leptin gene expression. The aim of this article was to study whether a high-energy diet may affect leptin gene promoter methylation in rats. A group of eleven male Wistar rats were assigned into two dietary groups, one fed on a control diet for 11 weeks and the other on a high-fat cafeteria diet. Rats fed a high-energy diet become overweight and hyperleptinemic as compared to the controls. DNA isolated from retroperitoneal adipocytes was treated with bisulfite and a distal portion of leptin promoter (from -694 to -372 bp) including 13 CpG sites was amplified by PCR and sequenced. The studied promoter portion was slightly more methylated in the cafeteria-fed animals, which was statistically significant (p < 0.05) for one of the CpG sites (located at the position -443). In obese rats, such methylation was associated to lower circulating leptin levels, suggesting that this position could be important in the regulation of leptin gene expression, probably by being a target sequence of different transcription factors. Our findings reveal, for the first time, that leptin methylation pattern can be influenced by diet-induced obesity, and suggest that epigenetic mechanisms could be involved in obesity by regulating the expression of important epiobesigenic genes.

La obesidad inducida por dieta alta en grasas modifica el patrón de metilación del promotor de la leptina en la rata / High fat diet-induced obesity modifies the methylation pattern of leptin promoter in rats

Leptin is an adipokine involved in body weight and food intake regulation whosepromoter region presents CpG islands that could be subject to dynamic methylation.This methylation process could be affected by environmental (e.g. diet) or endogenous(e.g., adipocyte differentiation, inflammation, hypoxia) factors, and could influenceadipocyte leptin gene expression. The aim of this article was to study whether ahigh-energy diet may affect leptin gene promoter methylation in rats. A group ofeleven male Wistar rats were assigned into two dietary groups, one fed on a controldiet for 11 weeks and the other on a high-fat cafeteria diet. Rats fed a high-energy dietbecome overweight and hyperleptinemic as compared to the controls. DNA isolatedfrom retroperitoneal adipocytes was treated with bisulfite and a distal portion of leptinpromoter (from -694 to -372 bp) including 13 CpG sites was amplified by PCRand sequenced. The studied promoter portion was slightly more methylated in thecafeteria-fed animals, which was statistically significant (p<0.05) for one of the CpGsites (located at the position –443). In obese rats, such methylation was associated tolower circulating leptin levels, suggesting that this position could be important in theregulation of leptin gene expression, probably by being a target sequence of differenttranscription factors. Our findings reveal, for the first time, that leptin methylationpattern can be influenced by diet-induced obesity, and suggest that epigenetic mechanismscould be involved in obesity by regulating the expression of important epiobesigenicgenes(AU)

La leptina es una adipoquina implicada en laregulación del peso corporal y la ingesta energéticacuya región promotora presenta islasCpG que podrían ser metiladas dinámicamente.Este proceso de metilación podría verseafectado por factores ambientales, como ladieta, o endógenos, como la diferenciación adipocitaria,inflamación o hipoxia, y podríainfluir en la expresión de leptina por parte delos adipocitos. El objetivo de este artículo esestudiar si una dieta alta en grasa podría afectara la metilación del promotor de la leptina enratas. Un grupo de once ratas Wistar machofue dividido en dos subgrupos, uno alimentadocon dieta control durante 11 semanas y el otrocon dieta alta en grasa (dieta de cafetería). Lasratas alimentadas con la dieta rica en grasa presentaronsobrepeso e hiperleptinemia. El ADNaislado de los adipocitos retroperitoneales fuetratado con bisulfito y una porción distal delpromotor de la leptina (de la base -694 a la -372), conteniendo 13 sitios CpG, fue amplificadapor PCR y secuenciada. Esta región delpromotor apareció ligeramente más metiladaen los animales alimentados con dieta de cafetería,lo cuál fue especialmente significativo (p<0,05) para uno de los sitios CpG (en la posición-443). En las ratas obesas, la metilación seasoció a una disminución de los niveles de leptinacirculante, lo que sugiere que esta posiciónpodría ser importante en la regulación de laexpresión génica de esta adipoquina, probablementepor ser una secuencia diana de diferen-tes factores de transcripción. Nuestros resultados,por primera vez, ponen de manifiesto queel patrón de metilación del promotor de la leptinapuede estar influido por la obesidad inducidapor la dieta, y sugieren que los mecanismosepigenéticos podrían estar implicados enla reciente pandemia de obesidad mediante laregulación de la expresión de importantesgenes epiobesigénicos(AU)

Ascorbic acid oral treatment modifies lipolytic response and behavioural activity but not glucocorticoid metabolism in cafeteria diet-fed rats.

AIM: To analyse the effects of vitamin C (VC), a potent dietary antioxidant, oral supplementation on body weight gain, behavioural activity, lipolytic response and glucocorticoid metabolism in the early stages of diet-induced overweight in rats. METHODS: Food intake, locomotive activity and faecal corticosterone were assessed during the 14 day trial period. After 2 weeks, the animals were sacrificed and the body composition, biochemical markers and lipolytic response from isolated adipocytes from retroperitoneal white adipose tissue were examined. RESULTS: The intake of a high-fat diet by rats induced a significant increase in body weight, adiposity and insulin resistance markers as well as a decrease in faecal corticosterone levels compared with standard diet-fed rats. Interestingly, the animals fed on the cafeteria diet showed a significant increase in the isoproterenol-induced lipolytic response in isolated adipocytes. Furthermore, this cafeteria-fed group showed a reduced locomotive behaviour than the control rats. On the other hand, oral VC supplementation in animals receiving the high-fat diet restored the cafeteria diet effect in some of the analysed variables such as final body weight and plasma insulin to control group levels. Remarkably, increases in locomotive behaviour and a significant decrease in the lipolytic response induced by isoproterenol on isolated adipocytes from animals treated with VC were observed. CONCLUSION: This work demonstrates that an oral ascorbic acid supplementation has direct effects on behavioural activity and on adipocyte lipolysis in early obesity stages in rats, which could indicate a protective short-term role of this vitamin against adiposity induced by chronic high-fat diet consumption.

Influence of dietary macronutrient composition on adiposity and cellularity of different fat depots in Wistar rats.

The aim of this study was to investigate the role of dietary macronutrient content on adiposity parameters and adipocyte hypertrophy/hyperplasia in subcutaneous and visceral fat depots from Wistar rats using combined histological and computational approaches. For this purpose, male Wistar rats were distributed into 4 groups and were assigned to different nutritional interventions: Control group (chow diet); high-fat group, HF (60% E from fat); high-fat-sucrose group, HFS (45% E from fat and 17% from sucrose); and high-sucrose group, HS (42% E from sucrose). At day 35, rats were sacrificed, blood was collected, tissues were weighed and fragments of different fat depots were kept for histological analyses with the new softwareAdiposoft. Rats fed with HF, HFS and HS diets increased significantly body weight and total body fat against Control rats, being metabolic impairments more pronounced on HS rats than in the other groups. Cellularity analyses usingAdiposoft revealed that retroperitoneal adipose tissue is histologically different than mesenteric and subcutaneous ones, in relation to bigger adipocytes. The subcutaneous fat pad was the most sensitive to the diet, presenting adipocyte hypertrophy induced by HF diet and adipocyte hyperplasia induced by HS diet. The mesenteric fat pad had a similar but attenuated response in comparison to the subcutaneous adipose tissue, while retroperitoneal fat pad only presented adipocyte hyperplasia induced by the HS diet intake after 35 days of intervention. These findings provide new insights into the role of macronutrients in the development of hyperplastic obesity, which is characterized by the severity of the clinical features. Finally, a new tool for analyzing histological adipose samples is presented.