Ameliorating high-fat diet-induced sperm and testicular oxidative damage by micronutrient-based antioxidant intervention in rats.

PURPOSE: Emerging evidence from rodent studies suggests that high-fat-diet (HFD)-induced obesity is characterized by increased oxidative damage in sperm and testis. However, interventions using micronutrient supplementation to mitigate oxidative damage in obesity have not been extensively studied. This study aimed to investigate the effect of an antioxidant-based micronutrient supplement (added folate, vitamin B6, choline, betaine, and zinc) on sperm and testicular oxidative damage in HFD-fed male Sprague Dawley rats. METHODS: Rats (3-weeks-old, 12/group) were weaned onto control (C) or HFD (H) or these diets with micronutrient supplement (CS; HS); sperm and testis were harvested at 30.5 weeks. To assess oxidative stress and antioxidant capacity in testis, levels of malondialdehyde (MDA), glutathione (GSH), folate and susceptibility index (SI) of pro-oxidative damage, mRNA expression of Nrf2, NFκB-p65, IL-6, IL-10 and TNF-α, in addition to superoxide-dismutase (SOD), catalase and glutathione-peroxidase (GPx) activities were measured. 8-hydroxy-2-deoxyguanosine (8-OHdG) were assessed in both sperm and testis. RESULTS: HFD-fed rats had significantly increased 8-OHdG content in sperm and testis, increased testicular SI, decreased testicular weight, SOD and GPx activity compared to control. Strikingly, supplementation of HFD appeared to significantly reduce 8-OHdG in sperm and testis (22% and 24.3%, respectively), reduce testicular SI and MDA content (28% and 40%, respectively), increase testicular weight (24%), SOD and GPX activity (30% and 70%, respectively) and GSH content (19%). Moreover, supplementation had significant impact to increase testicular folate content regardless of diet. Furthermore, an overall effect of supplementation to increase testicular mRNA expression of Nrf2 was observed across groups. Interestingly, testicular SI was positively correlated with sperm and testicular 8-OHdG and MDA content, suggesting a critical role of testicular antioxidant activity to combat oxidative damage in sperm and testis. CONCLUSION: Our findings suggest that antioxidant-based micronutrient supplement has the potential to interrupt HFD-induced sperm and testicular oxidative damage by improving testicular antioxidant capacity.

Effects of Micronutrient Supplementation on Glucose and Hepatic Lipid Metabolism in a Rat Model of Diet Induced Obesity.

Obesity increases the risk of metabolic disorders, partly through increased oxidative stress. Here, we examined the effects of a dietary micronutrient supplement (consisting of folate, vitamin B6, choline, betaine, and zinc) with antioxidant and methyl donor activities. Male Sprague Dawley rats (3 weeks old, 17/group) were weaned onto control (C) or high-fat diet (HFD) or same diets with added micronutrient supplement (CS; HS). At 14.5 weeks of age, body composition was measured by magnetic resonance imaging. At 21 weeks of age, respiratory quotient and energy expenditure was measured using Comprehensive Lab Animal Monitoring System. At 22 weeks of age, an oral glucose tolerance test (OGTT) was performed, and using fasting glucose and insulin values, Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) was calculated as a surrogate measure of insulin resistance. At 30.5 weeks of age, blood and liver tissues were harvested. Liver antioxidant capacity, lipids and expression of genes involved in lipid metabolism (Cd36, Fabp1, Acaca, Fasn, Cpt1a, Srebf1) were measured. HFD increased adiposity (p < 0.001) and body weight (p < 0.001), both of which did not occur in the HS group. The animals fed HFD developed impaired fasting glucose, impaired glucose tolerance, and fasting hyperinsulinemia compared to control fed animals. Interestingly, HS animals demonstrated an improvement in fasting glucose and fasting insulin. Based on insulin release during OGTT and HOMA-IR, the supplement appeared to reduce the insulin resistance developed by HFD feeding. Supplementation increased hepatic glutathione content (p < 0.05) and reduced hepatic triglyceride accumulation (p < 0.001) regardless of diet; this was accompanied by altered gene expression (particularly of CPT-1). Our findings show that dietary micronutrient supplementation can reduce weight gain and adiposity, improve glucose metabolism, and improve hepatic antioxidant capacity and lipid metabolism in response to HFD intake.

Cross-talk among metabolic parameters, esophageal microbiota, and host gene expression following chronic exposure to an obesogenic diet.

Unhealthy diets, and ensuing weight gain, predispose individuals to the development of esophageal adenocarcinoma. We examined the effect of chronic high fat diet (HFD) on the esophageal microbiota of Sprague Dawley rats using Illumina MiSeq amplicon sequencing (V4, 515 F/806 R) and on esophageal expression of IL18, PTGS2, PPARA, FFAR3, and CRAT. The relationships among metabolic parameters, esophageal microbiota, and host gene expression were determined. We observed a significant difference between the upper and lower esophageal microbiota in control fed rats, emphasized by enrichment of Lactobacillus species in the lower esophagus. Rats on HFD gained significantly more fat and had reduced insulin sensitivity. Diet type significantly affected the esophageal microbiota, with Clostridium sensu stricto being enriched in both upper and lower segments of HFD fed rats. Of interest, bacterial pathways related to carotenoid biosynthesis were significantly decreased in the lower esophagus of HFD fed rats. We observed strong correlations between metabolic parameters, the esophageal microbial profiles, and host esophageal gene expression. In particular, Fusobacterium, Rothia, and Granulicatella showed consistent correlations across a range of metabolic and gene markers. Our data indicates that unhealthy diets can significantly alter the esophageal microbiota, and enrich for bacterial species previously associated with chronic gastrointestinal diseases.

Effects of paternal obesity on growth and adiposity of male rat offspring.

Emerging evidence suggests that paternal obesity plays an important role in offspring health. Our previous work using a rodent model of diet-induced paternal obesity showed that female offspring from high-fat diet (HFD)-fed fathers develop glucose intolerance due to impairment of pancreatic insulin secretion. Here, we focused on the health outcomes of male offspring from HFD-fed fathers. Male Sprague-Dawley rats (3 wk old) were fed control (CD-F0) or HFD (HFD-F0) for 12 wk before mating with control-fed females. Male offspring were fed control diets for up to 8 wk or 6 mo. Although male offspring from HFD-F0 did not develop any obvious glucose metabolism defects in this study, surprisingly, a growth deficit phenotype was observed from birth to 6 mo of age. Male offspring from HFD-F0 had reduced birth weight compared with CD-F0, followed by reduced postweaning growth from 9 wk of age. This resulted in 10% reduction in body weight at 6 mo with significantly smaller fat pads and skeletal muscles. Reduced circulating levels of growth hormone (GH) and IGF-I were detected at 8 wk and 6 mo, respectively. Expression of adipogenesis markers was decreased in adipose tissue of HFD-F0 offspring at 8 wk and 6 mo, and expression of growth markers was decreased in muscle of HFD-F0 offspring at 8 wk. We propose that the reduced GH secretion at 8 wk of age altered the growth of male offspring from HFD-F0, resulting in smaller animals from 9 wk to 6 mo of age. Furthermore, increased muscle triglyceride content and expression of lipogenic genes were observed in HFD-F0 offspring, potentially increasing their metabolic risk.

Paternal High Fat Diet in Rats Leads to Renal Accumulation of Lipid and Tubular Changes in Adult Offspring.

Along with diabetes and obesity, chronic kidney disease (CKD) is increasing across the globe. Although some data support an effect of maternal obesity on offspring kidney, the impact of paternal obesity is unknown; thus, we have studied the effect of paternal obesity prior to conception. Male Sprague Dawley rats were fed chow diet or high fat diet (HFD) for 13-14 weeks before mating with chow-fed females. Male offspring were weaned onto chow and killed at 27 weeks for renal gene expression and histology. Fathers on HFD were 30% heavier than Controls at mating. At 27 weeks of age offspring of obese fathers weighed 10% less; kidney triglyceride content was significantly increased (5.35 ± 0.84 vs. 2.99 ± 0.47 µg/mg, p < 0.05, n = 8 litters per group. Histological analysis of the kidney demonstrated signs of tubule damage, with significantly greater loss of brush border, and increased cell sloughing in offspring of obese compared to Control fathers. Acat1, involved in entry of fatty acid for beta-oxidation, was significantly upregulated, possibly to counteract increased triglyceride storage. However other genes involved in lipid metabolism, inflammation and kidney injury showed no changes. Paternal obesity was associated with renal triglyceride accumulation and histological changes in tubules, suggesting a mild renal insult in offspring, who may be at risk of developing CKD.

Obesity-induced sperm DNA methylation changes at satellite repeats are reprogrammed in rat offspring.

There is now strong evidence that the paternal contribution to offspring phenotype at fertilisation is more than just DNA. However, the identity and mechanisms of this nongenetic inheritance are poorly understood. One of the more important questions in this research area is: do changes in sperm DNA methylation have phenotypic consequences for offspring? We have previously reported that offspring of obese male rats have altered glucose metabolism compared with controls and that this effect was inherited through nongenetic means. Here, we describe investigations into sperm DNA methylation in a new cohort using the same protocol. Male rats on a high-fat diet were 30% heavier than control-fed males at the time of mating (16-19 weeks old, n = 14/14). A small (0.25%) increase in total 5-methyl-2Ͳ-deoxycytidine was detected in obese rat spermatozoa by liquid chromatography tandem mass spectrometry. Examination of the repetitive fraction of the genome with methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) and pyrosequencing revealed that retrotransposon DNA methylation states in spermatozoa were not affected by obesity, but methylation at satellite repeats throughout the genome was increased. However, examination of muscle, liver, and spermatozoa from male 27-week-old offspring from obese and control fathers (both groups from n = 8 fathers) revealed that normal DNA methylation levels were restored during offspring development. Furthermore, no changes were found in three genomic imprints in obese rat spermatozoa. Our findings have implications for transgenerational epigenetic reprogramming. They suggest that postfertilization mechanisms exist for normalising some environmentally-induced DNA methylation changes in sperm cells.

Changes in gut microbiota in rats fed a high fat diet correlate with obesity-associated metabolic parameters.

The gut microbiota is emerging as a new factor in the development of obesity. Many studies have described changes in microbiota composition in response to obesity and high fat diet (HFD) at the phylum level. In this study we used 16s RNA high throughput sequencing on faecal samples from rats chronically fed HFD or control chow (n = 10 per group, 16 weeks) to investigate changes in gut microbiota composition at the species level. 53.17% dissimilarity between groups was observed at the species level. Lactobacillus intestinalis dominated the microbiota in rats under the chow diet. However this species was considerably less abundant in rats fed HFD (P<0.0001), this being compensated by an increase in abundance of propionate/acetate producing species. To further understand the influence of these species on the development of the obese phenotype, we correlated their abundance with metabolic parameters associated with obesity. Of the taxa contributing the most to dissimilarity between groups, 10 presented significant correlations with at least one of the tested parameters, three of them correlated positively with all metabolic parameters: Phascolarctobacterium, Proteus mirabilis and Veillonellaceae, all propionate/acetate producers. Lactobacillus intestinalis was the only species whose abundance was negatively correlated with change in body weight and fat mass. This species decreased drastically in response to HFD, favouring propionate/acetate producing bacterial species whose abundance was strongly correlated with adiposity and deterioration of metabolic factors. Our observations suggest that these species may play a key role in the development of obesity in response to a HFD.

Parental programming: how can we improve study design to discern the molecular mechanisms?

The contribution of inherited non-genetic factors to complex diseases is of great current interest. The ways in which mothers and fathers can affect their offspring's health clearly differ as a result of the intimate interactions between mother and offspring during pre- and postnatal life. There is, however, potential for some overlap in mechanisms, particularly epigenetic mechanisms. A small number of epidemiological studies and animal models have investigated the non-genetic contribution of the parents to offspring health. Discovering new mechanisms of disease inheritance is technically difficult, especially in genetically, socially and environmentally heterogeneous human populations. Therefore, rigorous experimental design, appropriate sample numbers and the use of high-throughput technologies are necessary to provide convincing evidence. Based on recent examples from the literature, here we propose several ways to improve human studies that aim to identify the underlying mechanisms of transgenerational inheritance of metabolic disease.

A new role for sterol regulatory element binding protein 1 transcription factors in the regulation of muscle mass and muscle cell differentiation.

The role of the transcription factors sterol regulatory element binding protein 1a (SREBP-1a) and SREBP-1c in the regulation of cholesterol and fatty acid metabolism has been well studied; however, little is known about their specific function in muscle. In the present study, analysis of recent microarray data from muscle cells overexpressing SREBP1 suggested that they may play a role in the regulation of myogenesis. We then demonstrated that SREBP-1a and -1c inhibit myoblast-to-myotube differentiation and also induce in vivo and in vitro muscle atrophy. Furthermore, we have identified the transcriptional repressors BHLHB2 and BHLHB3 as mediators of these effects of SREBP-1a and -1c in muscle. Both repressors are SREBP-1 target genes, and they affect the expression of numerous genes involved in the myogenic program. Our findings identify a new role for SREBP-1 transcription factors in muscle, thus linking the control of muscle mass to metabolic pathways.

Microarray analyses of SREBP-1a and SREBP-1c target genes identify new regulatory pathways in muscle.

In this study we have identified the target genes of sterol regulatory element binding protein (SREBP)-1a and SREBP-1c in primary cultures of human skeletal muscle cells, using adenoviral vectors expressing the mature nuclear form of human SREBP-1a or SREBP-1c combined with oligonucleotide microarrays. Overexpression of SREBP-1a led to significant changes in the expression of 1,315 genes (655 upregulated and 660 downregulated), whereas overexpression of SREBP-1c modified the mRNA level of 514 genes (310 upregulated and 204 downregulated). Gene ontology analysis indicated that in human muscle cells SREBP-1a and -1c are involved in the regulation of a large number of genes that are at the crossroads of different functional pathways, several of which are not directly connected with cholesterol and lipid metabolism. Six hundred fifty-two of all genes identified to be differentially regulated on SREBP overexpression had a sterol regulatory element (SRE) motif in their promoter sequences. Among these, 429 were specifically regulated by SREBP-1a, 69 by SREBP-1c, and 154 by both 1a and 1c. Because both isoforms recognize the same binding motif, we determined whether some of these functional differences could depend on the environment of the SRE motifs in the promoters. Results from promoter analysis showed that different combinations of transcription factor binding sites around the SRE binding motifs may determine regulatory networks of transcription that could explain the superposition of lipid and cholesterol metabolism with various other pathways involved in adaptive responses to stress like hypoxia and heat shock, or involvement in the immune response.