HF Diet Feeding Research Articles

High-fat diet during early life reshapes the gut microbiome and is associated with the disrupted mammary microenvironment in later life in mice

The influence of gut microbiota on gut health is well-documented, but it remains obscure for extraintestinal diseases such as breast cancer. Moreover, it is entirely unknown how gut dysbiosis during early life contributes to breast tumorigenesis later in life. In this study, we hypothesized that a high-fat diet during early life leads to alterations in the gut microbiome and is associated with disruptions in the mammary microenvironment. Female C57BL/6 mice were fed a low-fat diet (10% kcal fat) or a high-fat diet (HF, 60% kcal fat) for 8 weeks from the age of 4 to 12 weeks, which is equivalent to human childhood and adolescence. Twelve mice were sacrificed immediately after the 8-week feeding, the remainder were euthanized after switching to a normal lifecycle-supporting diet for an additional 12 weeks; the gut microbiome was then sequenced. The 8-week HF diet feeding altered the beta-diversity (Bray & Jaccard P < .01), and the difference remained significant after switching the diet (Bray & Jaccard P < .05). Immediately after HF feeding, a greater number of microbial taxa (>50) were altered, and about half of the taxa (25) remained significantly changed after switching the diet. The abundance of Alistipes, Bilophila, and Rikenellaceae stood out as significantly associated with multiple metabolic and inflammatory biomarkers in mammary tissue, including aromatase, Ccl2, and Cox2. In conclusion, an 8-week early-life HF feeding reshaped the gut microbiome, which connected with disrupted mammary microenvironments.

Impact of High-Fat Diet in Early-Life on Mammary Metabolic and Inflammatory Status in Later-Life in Mice

Abstract Objectives Emerging evidence indicates a potentially important role for early-life events and exposures in cancer development later in life. Moreover, accumulating evidence suggests that the incidence of cancers has reached a plateau in elders, whereas it continuously rises in young to middle adult. The present study aimed to investigate the potential impacts of high-fat diet in early-life, mimicking childhood/adolescent in humans, on mammary health in later-life of mice, equivalent to the young to middle age in human. Methods Female C57BL/8 mice (4 weeks of age) were fed a low-fat diet (LF: 10% kcal from fat) or a high-fat diet (HF: 60% kcal from fat) for 8 weeks, which is equivalent to child/adolescent age in humans. Mice in early-life groups were sacrificed after 8 weeks feeding, whereas mice in later-life groups were switched to standard chow diet (Lab Diet#5P76) and fed for additional 12 weeks before sacrifice. A panel of metabolic parameters, inflammatory cytokines, as well as gene expression related to tumorigenic Wnt-signaling were assessed by qPCR and immunoblotting analysis. Results Compared with LF group, the body weight in HF group was significantly elevated after 8-wk HF diet feeding (P &amp;lt; 0.05). After switching to the standard chow diet for 12 weeks, the significance remained until 24 weeks of age although with a reduced degree of magnitude (P &amp;lt; 0.05). For the metabolic factors, HFD reduced the expression levels of both Pparγ (P = 0.08) and adiponectin (P &amp;lt; 0.05) at 12 weeks and the reductions remains at 24 weeks (P &amp;lt; 0.01). Meanwhile, expressions of aromatase, estrogen receptor α and Tnf-α, Il-6, Il-10 as well as Cox2 among examined inflammatory mediators (Tnf-α, Il-6, Il-10, Il-2, Il-1β, Ifn-γ, Cox2) were significantly higher in HF than in LF group at 24 weeks (P &amp;lt; 0.05). For Wnt-signaling target genes (Cyclin D1, C-Myc, and Axin 2), a significant increase for C-Myc was observed in HF group at 12 weeks (P &amp;lt; 0.01). Conclusions Our results suggested that HF diet in early-life enhances adiposity and alters mammary metabolic and inflammatory status, creating a microenvironment in favor of breast tumorigenesis in later-life. Funding Sources This project was supported by USDA/Hatch (#1013548).

High protein intake promotes the adaptation to chronic hypoxia in zebrafish (Danio rerio)

Chronic hypoxia is a common environmental stress factor in aquaculture. Many researchers have illustrated the physiological and metabolic changes under chronic hypoxia, but there are few studies to evaluate the relationship between dietary nutrients and hypoxic adaptation in fish. In the present study, we fed zebrafish with control diet (C), high-carbohydrate diet (HC), high-fat diet (HF) and high-protein diet (HP) at normal dissolved oxygen (DO = 7.0 ± 0.5 mg/L) or low dissolved oxygen (DO = 2.5 ± 0.5 mg/L) environment, respectively. After a 7-week feeding trial, the growth performance, body composition, tissue biochemical indexes, mRNA expressions of hifα isoforms and nutrient metabolism-related genes, intestinal digestive enzyme activities and microbiota composition were measured. The results indicated that only the HP diet could alleviate the reduction of body weight and feed efficiency induced by chronic hypoxia, but the HF diet feeding worsened the adverse effects of chronic hypoxia in zebrafish. At the C groups, hypoxia increased catabolism of lipid and protein, but at the HP groups, hypoxia enhanced utilization of glycogen and protein. Compared to the C-hypoxia group, the HP feeding could activate transcription of hif-1a and its downstream glycolysis genes in chronic hypoxia. Furthermore, at hypoxic state, the HP feeding could alleviate the reduction of intestinal digestive enzyme activities and microbiota dysbiosis induced by the chronic hypoxia, including increasing bacterial diversity and decreasing Cetobacterium genus abundance. This is the first study indicating that high protein intake could strengthen ability of fish in resisting the negative effect of chronic hypoxia.

The gut bacterium Extibacter muris produces secondary bile acids and influences liver physiology in gnotobiotic mice

ABSTRACT Extibacter muris is a newly described mouse gut bacterium which metabolizes cholic acid (CA) to deoxycholic acid (DCA) via 7α-dehydroxylation. Although bile acids influence metabolic and inflammatory responses, few in vivo models exist for studying their metabolism and impact on the host. Mice were colonized from birth with the simplified community Oligo-MM12 with or without E. muris. As the metabolism of bile acids is known to affect lipid homeostasis, mice were fed either a low- or high-fat diet for eight weeks before sampling and analyses targeting the gut and liver. Multiple Oligo-MM12 strains were capable of deconjugating primary bile acids in vitro. E. muris produced DCA from CA either as pure compound or in mouse bile. This production was inducible by CA in vitro. Ursodeoxycholic, chenodeoxycholic, and β-muricholic acid were not metabolized under the conditions tested. All gnotobiotic mice were stably colonized with E. muris, which showed higher relative abundances after HF diet feeding. The presence of E. muris had minor, diet-dependent effects on Oligo-MM12 communities. The secondary bile acids DCA and surprisingly LCA and their taurine conjugates were detected exclusively in E. muris-colonized mice. E. muris colonization did not influence body weight, white adipose tissue mass, liver histopathology, hepatic aspartate aminotransferase, or blood levels of cholesterol, insulin, and paralytic peptide (PP). However, proteomics revealed shifts in hepatic pathways involved in amino acid, glucose, lipid, energy, and drug metabolism in E. muris-colonized mice. Liver fatty acid composition was substantially altered by dietary fat but not by E. muris.In summary, E. muris stably colonized the gut of mice harboring a simplified community and produced secondary bile acids, which affected proteomes in the liver. This new gnotobiotic mouse model can now be used to study the pathophysiological role of secondary bile acids in vivo.

Deletion of EHD2 Attenuated Adipocyte Hypertrophic Expansion and Ameliorated Diet-Induced Obesity and Insulin Resistance

Abstract Objectives Obesity induces adipocyte hypertrophy, which promotes metabolic dysfunction. The EPS15-Homology Domain-Containing (EHD) 2 is an endocytic traffic-regulatory protein regulating caveolae stability, thereby participating cell size expansion. Although EHD2 is abundantly expressed in adipocytes, very little information is available on its role on adipose metabolism. This study aimed to define the role of EHD2 on governing adipocyte size, lipid metabolism and insulin sensitivity. Methods To prepare the EHD2-deficient adipocytes, primary ear mesenchymal stem cells were isolate from wildtype (WT) and EHD2 KO mice and induced differentiation into adipocytes. To induce obesity and insulin resistance, EHD2 KO and WT male mice were fed a high-fat diet (50% calorie from lard) for 8 weeks. The changes in body weight was monitored weekly. Fasting plasma glucose and insulin levels were determined by glucometer and ELISA respectively. Glucose tolerance test (GTT) was conducted after HF diet feeding. To investigate insulin signaling, human recombinant insulin (1 U/kg BW) was injected peritoneally and epididymal fat was collected immediately for measuring the phosphorylation levels of AKT (p-AKT), a downstream target for insulin. To determine the lipogenic gene and protein expression, qPCR and Western blot analysis were conducted. Results Deletion of EHD2 markedly upregulated EHD1 expression in primary adipocytes. Also, deletion of EHD2 significantly attenuated adipocyte differentiation and maintained smaller lipid droplets. Consistently, absence of EHD2 was linked with reduced lipogenic gene expression. In vivo study, EHD2 KO mice exhibited slightly lower total body weight, but fat mass was markedly reduced. After 8 weeks of HF diet, EHD2 KO mice had lower levels of plasma glucose and insulin levels compared with WT. EHD2 KO mice were more glucose tolerant during GTT. Insulin signaling study revealed that EHD2 KO mice showed higher levels of insulin-stimulated p-AKT compared to WT mice, indicating EHD2 deletion promotes insulin sensitivity. Conclusions This study suggests that EHD2 is required for maximal adipocyte differentiation and hypertrophic expansion. The absence of EHD2 was linked with improved insulin and glucose sensitivity, presumably due to reduced adiposity and adipocyte size. Funding Sources Nebraska EPSCoR (Food for Health 2017) Seed Grant.

Free fatty acid receptor 3 differentially contributes to β-cell compensation under high-fat diet and streptozotocin stress.

The free fatty acid receptor 3 (FFA3) is a nutrient sensor of gut microbiota-generated nutrients, the short-chain fatty acids. Previously, we have shown that FFA3 is expressed in β-cells and inhibits islet insulin secretion ex vivo. Here, we determined the physiological relevance of the above observation by challenging wild-type (WT) and FFA3 knockout (KO) male mice with 1) hyperglycemia and monitoring insulin response via highly sensitive hyperglycemic clamps, 2) dietary high fat (HF), and 3) chemical-induced diabetes. As expected, FFA3 KO mice exhibited significantly higher insulin secretion and glucose infusion rate in hyperglycemic clamps. Predictably, under metabolic stress induced by HF-diet feeding, FFA3 KO mice exhibited less glucose intolerance compared with the WT mice. Moreover, similar islet architecture and β-cell area in HF diet-fed FFA3 KO and WT mice was observed. Upon challenge with streptozotocin (STZ), FFA3 KO mice initially exhibited a tendency for an accelerated incidence of diabetes compared with the WT mice. However, this difference was not maintained. Similar glycemia and β-cell mass loss was observed in both genotypes 10 days post-STZ challenge. Higher resistance to STZ-induced diabetes in WT mice could be due to higher basal islet autophagy. However, this difference was not protective because in response to STZ, similar autophagy induction was observed in both WT and FFA3 KO islets. These data demonstrate that FFA3 plays a role in modulating insulin secretion and β-cell response to stressors. The β-cell FFA3 and autophagy link warrant further research.

Gene expression modulation of lipid and central energetic metabolism related genes by high-fat diet intake in the main homeostatic tissues.

It is generally assumed that high-fat (HF) diets induce characteristic gene expression regulation, which may cause metabolic alterations associated with an increased risk of obesity, metabolic syndrome and other health alterations. However, an integrative analysis of the effects in different tissues is lacking, thus preventing new strategies to prevent or counteract permanent consequences. This review particularly focuses on lipid metabolism gene expression modulation in key homeostatic tissues in response to HF diet intake. The effects of HF diets on gene expression are mediated by insulin, leptin and nutrients such as fatty acids (FA) and glucose that alter insulin signalling. As a general trend, HF diet feeding induces the expression of catabolism related genes and reduces the expression of lipogenic related genes in the main homeostatic tissues. However, the effects of HF diets on gene expression depend on the amount and composition of fat, and there are specific effects in different animal models. From another angle, delayed effects associated with long-lasting changes caused by metabolic imprinting are of increasing interest. Finally, when considering the need for biomarkers of early metabolic effects, it is noticeable that many transcriptional adaptations that occur in tissues are reflected in peripheral blood mononuclear cells, which offers the possibility of using these minimally invasive samples in human studies.

Alterations of Gut Microbiota Prior to Changes of Paneth Cell‐antimicrobial Peptides in High Fat‐induced Obese Mice

It is under intensive argument about the reciprocal relationship between gut microbiota and the altered intestinal metabolic and inflammatory status in the obese state. Gut microbiota composition and Paneth cells is closely associated, especially in obesity. This study aimed to investigate the mutual interaction between the Paneth cell‐produced antimicrobial peptides expression and shift of the intestinal microbiota in obesity. Eight male C57BL/6 mice were fed a low fat diet (LF, 10%) as the control and 24 animals were fed a high fat diet (HF, 60%) and sacrificed sequentially with 8, 12, 16 weeks of feeding. The expression of antimicrobial peptides and the composition of gut microbiota were determined at each stage. The mice with 8‐wk HF feeding had higher fat percentage and significant changes of the gut microbiota comparing to the LF control group (p &lt; 0.05). The abundances of Firmicutes and Bacteroidetes from 8‐ to 16‐wk HF feeding were steady, with a higher abundance of Firmicutes and a lower of Bacteroidetes. However, 8‐wk HF diet feeding did not changed the Paneth antimicrobial peptides: lysozyme (LYZ), α‐defensin 5, and RegIIIγ (p &gt; 0.05), but reduced LYZ and RegIIIγ mRNA expression was observed in animals with 12‐ and 16‐wk HF feeding (p &lt; 0.05), and the results for LYZ protein level, measured by immunohistochemistry, also indicated a decrease after 16‐wk HF feeding. These results indicated that the high fat diet initially alters the profile of gut microbiota in obese mice, and changes in the expression of antimicrobial peptides occurs secondary to alterations in the composition of the intestinal microbiota.Support or Funding InformationThis project is supported by National Natural Science Foundation of China (Grant No.31401488), USDA grants (2014‐67017‐21762 and MAS00454), and Deans Incentive Grant.

Gestational Exposure to Maternal Obesity Influences β3‐Adrenergic Agonist Induced Beiging of White Adipose Tissue in Offspring

Maternal obesity is now recognized as an important contributing factor to the currently unfolding epidemic of obesity. In animal models, maternal obesity and exposure to high‐fat diets in utero, unequivocally predisposes the offspring to greater weight gain, metabolic dysfunction and a host of other related comorbidities, such as fatty liver disease. Our previous work in both rat and mouse models of obesity showed that offspring of obese dams are hyper‐responsive to an obesogenic challenge post‐weaning, and gain greater weight and fat mass. In part this is associated with decreased energy expenditure, impaired expression of mitochondrial OXPHOS and lipid oxidative genes and lower expression uncoupling proteins in liver, skeletal muscle and white adipose tissue. Nonetheless, the consequences of maternal obesity on programming of adipose tissue development, especially brown/beige adipose tissue development are unknown. In this report, we investigated role of maternal obesity on beige fat development in offspring following β3‐adrenergic agonist. Female C57BL6/J mice were fed diets with either low (10%) or HFD (45% of calories) for 12 wk starting at 5 wk of age. Dams were mated with males on control diets and provided access to respective diets during pregnancy and lactation. At weaning, offspring were weaned onto control and HFD, creating four groups (CC, CH, HC, HH, denoting maternal and offspring diets respectively). Male offspring from the four groups were followed till 20 wk of age. HFD‐fed offspring from HF dams (HH) gained significantly (p&lt;0.05) greater body weights than offspring from lean dams (CH). At 20 wk of age, offspring were treated with either vehicle or β3‐adrenergic receptor agonist CL‐316,243 (1 mg/kg, ip) for one week (N=4–5 each). CL‐316,243 is a well established activator of beige adipocyte formation. Twenty weeks of diet intervention lead to significantly consistent increased weight gain in HH compared to other groups. One week of CL injection towards to end of study did not affect the weight gain. There were no significant differences in overall body weight between vehicle and CL treated animals. Gene expression analysis of inguinal adipose tissue depot (iWAT) revealed significant differences in vehicle and CL treated groups. Expression of brown‐fat specific genes such as Ucp1, Cidea and Pgc1a was affected by maternal HF diet feeding in both vehicle and CL treated group. Moreover, maternal obesity hampered the response to CL‐316,243 consistent with programming defect in adipose tissue. Further analysis of developmental aspects of beiging of adipose tissue is under investigation. Our findings indicate that maternal obesity influences beige cell formation in response to classical beiging inducers which might influence modulation of energy expenditure, metabolism and overall risk of obesity.Support or Funding InformationSupport USDA CRIS 6206‐51000‐010‐05S

High‐fat diet feeding induces sex‐dependent changes in inflammatory and insulin sensitivity profiles of rat adipose tissue

The aim of the study was to determine, in rats of both sexes, the effect of HF diet feeding on the expression of adipokines involved in inflammatory status and insulin sensitivity and on the levels of proteins involved in lipid handling of retroperitoneal adipose tissue. Eight-week-old Wistar rats of both sexes were fed a control diet (2.9% w/w fat) or an HF diet (30% w/w fat) for 14 weeks. Adiponectin, peroxisome proliferator-activated receptor γ and inflammatory marker mRNA levels were analyzed by real-time polymerase chain reaction. Levels of insulin receptor, glucose transporter 4, carnitine palmitoyltransferase 1, fatty acid synthase, hormone-sensitive lipase and lipoprotein lipase were determined by Western blot. HF diet feeding did not induce hyperphagia or body weight gain but did promote an increase in adiposity although only in male rats. HF diet impaired glucose tolerance and the expression of inflammatory and insulin sensitivity markers in adipose tissue of male rats, but not in female rats. Male rats seem to be more prone to disorders associated with an unbalanced composition of the diet, even in the absence of hyperphagia. In contrast, female rats counteract excessive fat intake by improving their ability to use lipid fuels, which limits adiposity and maintains insulin sensitivity.

Leptin intake during the suckling period improves the metabolic response of adipose tissue to a high-fat diet

The intake of leptin during the suckling period protects against obesity and improves insulin and central leptin sensitivity in adult rats. We analyzed whether leptin treatment to neonates may also improve later peripheral leptin sensitivity in adipose tissue under high-fat (HF) diet conditions. Male rats were supplemented with a daily oral dose of leptin or the vehicle (controls) during the suckling period. After weaning, animals were fed a normal-fat or an HF diet until the age of 6 months. At this age, mRNA and protein levels of the long-form leptin receptor (OB-Rb) and the expression of other genes related with energy metabolism were measured in various adipose depots (inguinal, mesenteric and retroperitoneal). HF-diet feeding resulted in lower OB-Rb mRNA and protein levels in internal depots in controls but not in leptin-treated animals; these animals maintained OB-Rb mRNA and protein levels under HF-diet conditions in these depots, particularly in the mesenteric one, and this was accompanied by increased expression of genes related with energy uptake (GLUT4, CD36), fatty acid oxidation (peroxisome proliferator activated receptor-alpha (PPARalpha), CPT1, UCP3) and lipogenesis (PPARgamma, GPAT). Leptin-treatment also ameliorated HF-diet-induced hepatic fat accumulation occurring in control animals. Leptin treatment during the suckling period may improve the lasting effects of HF-diet feeding on leptin receptor abundance in the adipose tissue and increase its oxidative capacity, resulting in a better handling and partitioning of excess fuel. This, together with the described improvement of central leptin sensitivity, may explain why these animals are more protected against diet-induced obesity and its metabolic-related disorders.

Effect of high-fat diet feeding on leptin receptor expression in white adipose tissue in rats: depot- and sex-related differential response

Previous studies have illustrated the importance of leptin receptor (OB-Rb) mediated action on adipocytes in the regulation of body weight. The aim of the present study was to investigate in male and female rats the effects of high-fat (HF) diet feeding on the expression levels of OB-Rb in different depots of white adipose tissue (WAT), and its relation to fatty acid oxidation capacity. Male and female Wistar rats were fed until the age of 6 months with a normal-fat (NF) or non-isocaloric HF-diet (10 and 45% calories from fat, respectively). At this age, the weight of three different fat depots (retroperitoneal, mesenteric and inguinal) and the expression levels of OB-Rb, PPARalpha and CPT1 in these depots were measured. HF-diet feeding resulted in an increase in the weight of the different fat depots, the retroperitoneal depot being the one with the greatest increase in both sexes. In this depot, HF-diet feeding resulted in a significant decrease in OB-Rb mRNA levels, more marked in male than in female rats. In the mesenteric depot, the effects of HF-diet feeding on OB-Rb mRNA levels were sex-dependent: they decreased in males rats (associated with a decrease in PPARalpha and CPT1 mRNA levels), but increased in female rats. In the inguinal depot, OB-Rb expression was not affected by HF-diet feeding. These results show that a chronic intake of an HF-diet altered the expression of OB-Rb in WAT in a depot and sex-dependent manner. The decreased expression of OB-Rb in the internal depots of male rats under HF-diet feeding, with the resulting decrease in leptin sensitivity, can help to explain the higher tendency of males to suffer from obesity-linked disorders under HF-diet conditions.

Effects of high dietary fat and cholesterol on expression of PPARα, LXRα, and their responsive genes in the liver of apoE and LDLR double deficient mice

The significance of transcription factors PPAR alpha, LXR alpha, and their responsive/target genes for the pathogenesis of atherosclerosis in apolipoprotein E and low-density lipoprotein receptor double deficient (AL) mice fed with high fat and cholesterol (HF) diet were studied. C57BL/6J wild-type (WT) mice were used as control to the AL mice. Plasma lipid metabolites and morphological atherosclerotic lesions in aortic wall were determined. Semi- and real-time quantitative RT-PCR were used to measure gene expression patterns between AL mice and the controls, which were fed with HF or normal chow diet. The results showed that in AL mice fed with HF diet, plasma lipid levels, hepatic lipid accumulation, and atherogenesis together with upregulated PPAR alpha, LXR alpha, and their target genes, i.e., FAT, SCD1, FAS, Angptl3, and apoB100 significantly increased in a 12-week long feeding period. In contrast, apoAI, apoAIV, apoF, LPL, and SR-BI were decreased compared to chow-fed group. In WT mice, PPAR alpha, LXR alpha, FAS, Angpt13, CPT1, apoF, ACOX1, LPL, and SR-BI were increased with HF treatment, while apoAI and apoAIV were decreased markedly. The different changes of lipid metabolism-related genes between AL and WT mice, fed with HF diet or chow diet indicated that the mechanisms of dietary effects on gene mutant mice are different from those of intact WT mice. Since lipid metabolic system defected genetically in AL mice, we suggest that the changes of PPAR alpha, LXR alpha, and their target genes aggravated lipid metabolic disorder in the liver and further accelerated the development of atherosclerosis on a stress of HF diet feeding in AL mice.

Sex-associated differences in the leptin and ghrelin systems related with the induction of hyperphagia under high-fat diet exposure in rats

Leptin and ghrelin are known to be main hormones involved in the control of food intake, with opposing effects. Here we have explored whether changes in the leptin and ghrelin system are involved in the long-term effects of high-fat (HF) diet feeding in rats and whether sex-associated differences exist. Male and female Wistar rats were fed until the age of 6 months with a normal-fat (NF) or an HF-diet. Food intake and body weight were followed. Gastric and serum levels of leptin and ghrelin, and mRNA levels of leptin (in stomach and adipose tissue), ghrelin (in stomach), and NPY, POMC, and leptin and ghrelin receptors (OB-Rb and GHS-R) (in the hypothalamus) were measured. In both males and females, total caloric intake and body weight were greater under the HF-diet feeding. In females, circulating ghrelin levels and leptin mRNA expression in the stomach were higher under HF-diet. HF-diet feeding also resulted in higher hypothalamic NPY/POMC mRNA levels, more marked in females, and in lower OB-Rb mRNA levels, more marked in males. In addition, in females, serum ghrelin levels correlated positively with hypothalamic NPY mRNA levels, and these with caloric intake. In males, hypothalamic OB-Rb mRNA levels correlated positively with POMC mRNA levels and these correlated negatively with caloric intake and with body weight. These data reflect differences between sexes in the effects of HF-diet feeding on food intake control systems, suggesting an impairment of the anorexigenic leptin–POMC system in males and an over-stimulation of the orexigenic ghrelin–NPY system in females.