Intermittent fasting promotes rejuvenation of immunosenescent phenotypes in aged adipose tissue.

The aging of white adipose tissue (WAT) involves senescence of adipose stem and progenitor cells (ASPCs) and dysregulation of immune cell populations, serving as a major driver of age-associated adipose dysfunction and metabolic diseases. Conversely, the elimination of senescent ASPCs is associated with improvements in overall health. Intermittent fasting (IF), a dietary intervention that incorporates periodic cycles of fasting and refeeding, has been reported to promote weight loss and fat mass reduction and improve glucose and insulin homeostasis in both murine and human studies. While previous studies have assessed the effects of IF on obesity-associated metabolic dysfunction, few studies have examined the aging-specific changes to ASPCs and immune cell populations in WAT. Here, we show that IF in 18-20-month-old mice reduced senescent phenotypes of ASPCs and restored their adipogenic potential. Intriguingly, IF-treated mice exhibited an increase in adipose eosinophils, which has been reported to be associated with improved WAT homeostasis and immunological fitness in aged mice. The observed cellular and metabolic changes suggest that IF may be a feasible lifestyle regimen to reduce cellular senescence which could result in attenuation of downstream aging-induced WAT dysfunction and metabolic diseases.

The hedonic overdrive model best explains high-fat diet-induced obesity in C57BL/6 mice.

OBJECTIVE: High-fat diets cause obesity in male mice; however, the underlying mechanisms remain controversial. Here, three contrasting ideas were assessed: hedonic overdrive, reverse causality, and passive overconsumption models. METHODS: A total of 12 groups of 20 individually housed 12-week-old C57BL/6 male mice were exposed to 12 high-fat diets with varying fat content from 40% to 80% (by calories), protein content from 5% to 30%, and carbohydrate content from 8.4% to 40%. Body weight and food intake were monitored for 30 days after 7 days at baseline on a standard low-fat diet. RESULTS: After exposure to the diets, energy intake increased first, and body weight followed later. Intake then declined. The peak energy intake was dependent on both dietary protein and carbohydrate, but not the dietary fat and energy density, whereas the rate of decrease in intake was only related to dietary protein. On high-fat diets, the weight of food intake declined, but despite this average reduction of 14.4 g in food intake, they consumed, on average, 357 kJ more energy than at baseline. CONCLUSIONS: The hedonic overdrive model fit the data best. The other two models were not supported.

The Effects of Graded Levels of Calorie Restriction: XX. Impact of Long-Term Graded Calorie Restriction on Survival and Body Mass Dynamics in Male C57BL/6J Mice.

Calorie restriction (CR) typically promotes a reduction in body mass, which correlates with increased lifespan. We evaluated the overall changes in survival, body mass dynamics, and body composition following long-term graded CR (580 days/19 months) in male C57BL/6J mice. Control mice (0% restriction) were fed ad libitum in the dark phase only (12-hour ad libitum [12AL]). CR groups were restricted by 10%-40% of their baseline food intake (10CR, 20CR, 30CR, and 40CR). Body mass was recorded daily, and body composition was measured at 8 time points. At 728 days/24 months, all surviving mice were culled. A gradation in survival rate over the CR groups was found. The pattern of body mass loss differed over the graded CR groups. Whereas the lower CR groups rapidly resumed an energy balance with no significant loss of fat or fat-free mass, changes in the 30 and 40CR groups were attributed to higher fat-free mass loss and protection of fat mass. Day-to-day changes in body mass were less variable under CR than for the 12AL group. There was no indication that body mass was influenced by external factors. Partial autocorrelation analysis examined the relationship between daily changes in body masses. A negative correlation between mass on Day 0 and Day +1 declined with age in the 12AL but not the CR groups. A reduction in the correlation with age suggested body mass homeostasis is a marker of aging that declines at the end of life and is protected by CR.

High dietary protein and fat contents exacerbate hepatic senescence and SASP in mice.

Dietary choices have a profound impact on the aging process. In addition to the total amount of energy intake, macronutrient composition influences both health and lifespan. However, the exact mechanisms by which dietary macronutrients influence onset and progression of age-associated features remain poorly understood. Cellular senescence is a state of stable growth arrest characterized by the secretion of numerous bioactive molecules with pro-inflammatory properties. Accumulation of senescent cells is considered one of the basic mechanisms of aging and an important contributor to chronic inflammation and tissue degeneration. Whether dietary macronutrients affect the accumulation and the phenotype of senescent cells with age is still unknown. Here, we show that feeding on diets with varying ratios of dietary macronutrients for 3 months has a significant effect on different senescence-associated markers in the mouse liver. High protein intake is associated with higher expression levels of the two classical senescence-associated growth arrest genes, p21 and p16. Furthermore, the expression of many pro-inflammatory secretory markers was increased in diets enriched in protein and further enhanced by increases in fat content. These results provide preliminary evidence that dietary macronutrients have a significant influence on senescence markers and merit further investigation.

A single nucleotide mutation in the dual-oxidase 2 (DUOX2) gene causes some of the panda's unique metabolic phenotypes.

The giant panda (Ailuropoda melanoleuca) is an iconic bear native to China, famous for eating almost exclusively bamboo. This unusual dietary behavior for a carnivore is enabled by several key adaptations including low physical activity, reduced organ sizes and hypothyroidism leading to lowered energy expenditure. These adaptive phenotypes have been hypothesized to arise from a panda-unique single-nucleotide mutation in the dual-oxidase 2 (DUOX2) gene, involved in thyroid hormone synthesis. To test this hypothesis, we created genome-edited mice carrying the same point mutation as the panda and investigated its effect on metabolic phenotype. Homozygous mice were 27% smaller than heterozygous and wild-type ones, had 13% lower body mass-adjusted food intake, 55% decreased physical activity, lower mass of kidneys (11%) and brain (5%), lower serum thyroxine (T4: 36%), decreased absolute (12%) and mass-adjusted (5%) daily energy expenditure, and altered gut microbiota. Supplementation with T4 reversed the effects of the mutation. This work uses a state-of-the-art genome editing approach to demonstrate the link between a single-nucleotide mutation in a key endocrine-related gene and profound adaptive changes in the metabolic phenotype, with great importance in ecology and evolution.

Effects of dietary macronutrients on the hepatic transcriptome and serum metabolome in mice.

Dietary macronutrient composition influences both hepatic function and aging. Previous work suggested that longevity and hepatic gene expression levels were highly responsive to dietary protein, but almost unaffected by other macronutrients. In contrast, we found expression of 4005, 4232, and 4292 genes in the livers of mice were significantly associated with changes in dietary protein (5%-30%), fat (20%-60%), and carbohydrate (10%-75%), respectively. More genes in aging-related pathways (notably mTOR, IGF-1, and NF-kappaB) had significant correlations with dietary fat intake than protein and carbohydrate intake, and the pattern of gene expression changes in relation to dietary fat intake was in the opposite direction to the effect of graded levels of caloric restriction consistent with dietary fat having a negative impact on aging. We found 732, 808, and 995 serum metabolites were significantly correlated with dietary protein (5%-30%), fat (8.3%-80%), and carbohydrate (10%-80%) contents, respectively. Metabolomics pathway analysis revealed sphingosine-1-phosphate signaling was the significantly affected pathway by dietary fat content which has also been identified as significant changed metabolic pathway in the previous caloric restriction study. Our results suggest dietary fat has major impact on aging-related gene and metabolic pathways compared with other macronutrients.

Effect of disrupted episodic memory on food consumption: no impact of neuronal loss of endophilin A1 on food intake and energy balance.

Food intake is generally assumed to reflect a regulatory tension between homeostatic and hedonic drivers. Information from individuals with memory dysfunction suggests that episodic memory may also play a significant role. We reasoned that if memory influences food intake, then disrupting a genetic factor that is important in episodic memory formation should affect food intake and energy balance. We performed spatial learning tests on neuronal specific endophilin A1 (EENA1) KO mice using the four-arm baited version of the radial arms maze (RAM). Energy regulation has also been evaluated. As anticipated neuronal EENA1 KO mice had impaired spatial memory. However, loss of endophilin A1 did not result in greater food intake, or altered energy absorption efficiency, relative to wild-type (WT) mice, when fed either low or high fat diets. Moreover, loss of EENA1 did not significantly affect other features of energy balance-physical activity and energy expenditure. No statistically significant changes were observed in the expression of hypothalamic neuropeptides related to food intake regulation, or circulating levels of leptin. We conclude that food intake and energy balance are largely governed by homeostatic and hedonic processes, and when these processes are intact memory probably plays a relatively minor role in food intake regulation.

Impact of graded maternal dietary fat content on offspring susceptibility to high-fat diet in mice.

OBJECTIVE: Maternal high-fat diet (HFD) increases offspring obesity, yet the impacts of different levels of maternal dietary fat have seldom been addressed. In mice, the impact of graded maternal dietary fat on offspring adiposity and offspring's later susceptibility to HFD were assessed. METHODS: Lactating mice were fed diets with graded fat content from 8.3% to 66.6%. One male and one female pup from each litter were weaned onto a low-fat diet for 15 weeks. HFD (41.7%) was then introduced to half of the offspring for 12 weeks. RESULTS: Offspring body weight and adiposity were positively related to maternal dietary fat content and were higher when mothers were exposed to HFD. The maternal diet effect was nonlinear and sex dependent. A maternal dietary fat of 41.7% and above exaggerated the offspring body weight gain in males but was not significant in females. Maternal 8.3% fat and 25% fat diets led to the highest daily energy expenditure and respiratory exchange ratio in offspring. Offspring fed a low-fat diet had higher daily energy expenditure and respiratory exchange ratio than those fed an HFD. CONCLUSIONS: Increasing maternal dietary fat during lactation, and HFD in later life, had significant and interacting impacts on offspring obesity. Maternal diet had a bigger impact on male offspring. The effects of maternal dietary fat content were nonlinear.

Effects of dietary macronutrients and body composition on glucose homeostasis in mice.

As a major health issue, obesity is linked with elevated risk of type 2 diabetes. However, whether disrupted glucose homeostasis is due to altered body composition alone, or whether dietary macronutrients play an additional role, independent of their impact on body composition, remains unclear. We investigated the associations between macronutrients, body composition, blood hormones and glucose homeostasis. We fed C57BL/6N mice 29 different diets with variable macronutrients for 12 weeks. After 10 weeks, intraperitoneal glucose tolerance tests were performed. Generalized linear models were generated to evaluate the impacts of macronutrients, body composition and blood hormones on glucose homeostasis. The area under the glucose curve (AUC) was strongly associated with body fat mass, but not dietary macronutrients. AUC was significantly associated with fasting insulin levels. Six genes from transcriptomic analysis of epididymal white adipose tissue and subcutaneous white adipose tissue were significantly associated with AUC. These genes may encode secreted proteins that play important previously unanticipated roles in glucose homeostasis.

Surviving winter on the Qinghai-Tibetan Plateau: Pikas suppress energy demands and exploit yak feces to survive winter.

The Qinghai-Tibetan Plateau, with low precipitation, low oxygen partial pressure, and temperatures routinely dropping below -30 °C in winter, presents several physiological challenges to its fauna. Yet it is home to many endemic mammalian species, including the plateau pika (Ochotona curzoniae). How these small animals that are incapable of hibernation survive the winter is an enigma. Measurements of daily energy expenditure (DEE) using the doubly labeled water method show that pikas suppress their DEE during winter. At the same body weight, pikas in winter expend 29.7% less than in summer, despite ambient temperatures being approximately 25 °C lower. Combined with resting metabolic rates (RMRs), this gives them an exceptionally low metabolic scope in winter (DEE/RMRt = 1.60 ± 0.30; RMRt is resting metabolic rate at thermoneutrality). Using implanted body temperature loggers and filming in the wild, we show that this is achieved by reducing body temperature and physical activity. Thyroid hormone (T3 and T4) measurements indicate this metabolic suppression is probably mediated via the thyroid axis. Winter activity was lower at sites where domestic yak (Bos grunniens) densities were higher. Pikas supplement their food intake at these sites by eating yak feces, demonstrated by direct observation, identification of yak DNA in pika stomach contents, and greater convergence in the yak/pika microbiotas in winter. This interspecific coprophagy allows pikas to thrive where yak are abundant and partially explains why pika densities are higher where domestic yak, their supposed direct competitors for food, are more abundant.

The carbohydrate-insulin model does not explain the impact of varying dietary macronutrients on the body weight and adiposity of mice.

OBJECTIVES: The carbohydrate-insulin model (CIM) predicts that increases in fasting and post-prandial insulin in response to dietary carbohydrates stimulate energy intake and lower energy expenditures, leading to positive energy balance and weight gain. The objective of the present study was to directly test the CIM's predictions using C57BL/6 mice. METHODS: Diets were designed by altering dietary carbohydrates with either fixed protein or fat content and were fed to C57BL/6 mice acutely or chronically for 12 weeks. The body weight, body composition, food intake, and energy expenditures of the mice were measured. Their fasting and post-prandial glucose and insulin levels were also measured. RNA-seq was performed on RNA from the hypothalamus and subcutaneous white adipose tissue. Pathway analysis was conducted using IPA. RESULTS: Only the post-prandial insulin and fasting glucose levels followed the CIM's predictions. The lipolysis and leptin signaling pathways in the sWAT were inhibited in relation to the elevated fasting insulin, supporting the CIM's predicted impact of high insulin. However, because higher fasting insulin was unrelated to carbohydrate intake, the overall pattern did not support the model. Moreover, the hypothalamic hunger pathways were inhibited in relation to the increased fasting insulin, and the energy intake was not increased. The browning pathway in the sWAT was inhibited at higher insulin levels, but the daily energy expenditure was not altered. CONCLUSIONS: Two of the predictions were partially supported (and hence also partially not supported) and the other three predictions were not supported. We conclude that the CIM does not explain the impact of dietary macronutrients on adiposity in mice.

Antimicrobial capacity of ultrasound and ozone for enhancing bacterial safety on inoculated shredded green cabbage (Brassica oleracea var. capitata).

The high frequency and incidence of foodborne outbreaks related to fresh vegetables consumption is a major public health concern and an economic burden worldwide. This study evaluated the effect of individual and combined application of ultrasound (40 kHz, 100 W) and ozone on the inactivation of foodborne Escherichia coli and Salmonella, as well as their impact on cabbage color and vitamin C content. Plate count, scanning electron microscopy (SEM), and flow cytometry (FCM) following single or double staining with carboxyfluorescein diacetate and (or) propidium iodide were used to determine bacterial inactivation parameters, such as cell culturability, membrane integrity, intracellular enzyme activity, and injured and dead cells. The results of FCM and SEM showed that ultrasound treatment affected bacteria mainly by acting on the cell membrane and inactivating intracellular esterase, which resulted in bacterial death. Furthermore, when combined with ozone at 1.5 mg/L, the maximum reduction of bacterial populations was observed at 8 min with no damage on the surface of treated leaves. Therefore, fresh products sanitization using a combination of ultrasound and ozone has the potential to be an alternative for maintaining the color and vitamin C content of green cabbage.

Impact of dietary sucrose on adiposity and glucose homeostasis in C57BL/6J mice depends on mode of ingestion: liquid or solid.

OBJECTIVE: Although it is widely accepted that obesity results from an imbalance of energy intake and expenditure, the mechanisms underlying this process and effective strategies for prevention and treatment are unclear. Growing evidence suggests excess consumption of sugar may play an important role, yet we showed previously in mice that consuming up to 30% of calories as sucrose in the diet had no impact on weight regulation. Since in humans consumption of sugar-sweetened beverages has been widely implicated, we investigated whether the mode of ingestion (solid or liquid) had different impacts on body weight regulation and glucose homeostasis. METHODS: Dietary sucrose was delivered in solid (as part of a standard pelleted rodent chow) and liquid (in drinking water) to C57BL/6 mice for 8 weeks. Body weight, body composition, energy intake and expenditure were monitored, as well as glucose and insulin tolerance tests. Expression of sweet taste receptors on the tongue, and glycogen and fat contents of the liver were also measured. RESULTS: Consumption of sucrose-sweetened water, but not equivalent levels of solid sucrose, led to body fat gain in C57BL/6 mice. Glucose intolerance was positively correlated to body fatness, rather than sucrose intake. CONCLUSIONS: Our data support the suggestion that consumption of liquid sucrose may be an important contributor to dysregulation of body weight and related metabolic syndromes.

Dietary Fat, but Not Protein or Carbohydrate, Regulates Energy Intake and Causes Adiposity in Mice.

The impacts of different macronutrients on body weight regulation remain unresolved, with different studies suggesting increased dietary fat, increased carbohydrates (particularly sugars), or reduced protein may all stimulate overconsumption and drive obesity. We exposed C57BL/6 mice to 29 different diets varying from 8.3% to 80% fat, 10% to 80% carbohydrate, 5% to 30% protein, and 5% to 30% sucrose. Only increased dietary fat content was associated with elevated energy intake and adiposity. This response was associated with increased gene expression in the 5-HT receptors, and the dopamine and opioid signaling pathways in the hypothalamus. We replicated the core findings in four other mouse strains (DBA/2, BALB/c, FVB, and C3H). Mice regulate their food consumption primarily to meet an energy rather than a protein target, but this system can be over-ridden by hedonic factors linked to fat, but not sucrose, consumption.

VEGF and VEGFB Play Balancing Roles in Adipose Differentiation, Gene Expression, and Function.

Obesity is the result of abnormal adipose development and energy metabolism. Using vascular endothelial growth factor (VEGF) B-knockout and inducible VEGF downregulation mouse models, we have shown that VEGFB inactivation caused expansion of white adipose, whitening of brown adipose, an increase in fat accumulation, and a reduction in energy consumption. At the same time, expression of the white adipose-associated genes was increased and brown adipose-associated genes decreased. VEGF repression, in contrast, induced brown adipose expansion and brown adipocyte development in white adipose, increased energy expenditure, upregulated brown adipose-associated genes, and downregulated white adipose-associated genes. When VEGFB-knockout and VEGF-repressed mice are crossed together, VEGF and VEGFB can counteractively regulate large numbers of genes and efficiently reverse each other's roles. These genes, under counteractive VEGF and VEGFB regulations, include transcription factors, adhesion molecules, and metabolic enzymes. This balancing role is confirmed by morphologic and functional changes. This study reports that VEGF and VEGFB counteractively regulate adipose development and function in energy metabolism.

Expression Patterns and Potential Biological Roles of Dip2a.

Disconnected (disco)-interacting protein 2 homolog A is a member of the DIP2 protein family encoded by Dip2a gene. Dip2a expression pattern has never been systematically studied. Functions of Dip2a in embryonic development and adult are not known. To investigate Dip2a gene expression and function in embryo and adult, a Dip2a-LacZ mouse model was generated by insertion of ß-Gal cDNA after Dip2a promoter using CRISPR/Cas9 technology. Dip2a-LacZ mouse was designed to be a lacZ reporter mouse as well as a Dip2a knockout mouse. Heterozygous mice were used to study endogenous Dip2a expression and homozygotes to study DIP2A-associated structure and function. LacZ staining indicated that Dip2a is broadly expressed in neuronal, reproductive and vascular tissues, as well as in heart, kidney, liver and lung. Results demonstrate that Dip2a is expressed in ectoderm-derived tissues in developing embryos. Adult tissues showed rich staining in neurons, mesenchymal, endothelial, smooth muscle cells and cardiomyocytes by cell types. The expression pattern highly overlaps with FSTL1 and supports previous report that DIP2A to be potential receptor of FSTL1 and its protective roles of cardiomyocytes. Broad and intense embryonic and adult expression of Dip2a has implied their multiple structural and physiological roles.

Large genomic fragment deletions and insertions in mouse using CRISPR/Cas9.

ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and ß-galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies.