Mice Regulate Dietary Amino Acid Balance and Energy Intake by Selecting between Complementary Protein Sources.

BACKGROUND: A balanced intake of protein and constituent amino acids (AAs) requires adjustments to total food intake (protein leverage [PL]) and food selection to balance deficits and excesses (complementary feeding). We provided mice with choices of casein and whey, 2 protein sources that are complementary in AA balance, across a range of protein concentrations (P%) of digestible energy (DE). OBJECTIVES: We aimed to determine if: 1) PL operates similarly for casein and whey; 2) one protein source is preferred at control P%; 3) the preference changes as P% falls; and 4) AA intakes under control and low P% levels identify AAs that drive changes in protein selection. METHODS: Food intake and plasma fibroblast growth factor-21 (FGF21) concentrations were measured in mice at various P% (P7.5%-P33%). For direct comparisons, defined diets were used in which the protein source was either casein or whey. In food choice studies, mice had access to foods in which both casein and whey were provided at the same P% level at the same time. RESULTS: PL operated at different P% thresholds in casein (13%)- and whey (10%)-based diets, and the magnitude of PL was greater for casein. Although mice preferred casein under control conditions (P23%), a pronounced preference shift to whey occurred as P% fell to P13% and P10%. At low P%, increases in food intake were accompanied by increases in plasma FGF21, a protein hunger signal. Among AAs deficient in casein and enriched in whey, the intake of Cys was the most invariant as P% changed between P23% and P10%, appearing to drive the switch in protein preference. CONCLUSIONS: Mice selected between complementary protein sources, casein and whey, achieving stable total energy intake and regulated intake of AAs as P% varied. Supplementation of low P% casein diets with one whey-enriched AA, Cys, suppressed plasma FGF21 and total food intake.

Meta-analysis links dietary branched-chain amino acids to metabolic health in rodents.

BACKGROUND: The role of dietary branched chain amino acids (BCAAs) and their effect on metabolic health is complex. How dietary BCAA levels and their interaction with background nutrition affect health is unclear. Here, we used meta-analysis and meta-regression, together with the nutritional modelling, to analyse the results of rodent studies that increased the level of dietary BCAAs and measured circulating levels, outcomes related to metabolic health, body mass and food intake. RESULTS: Across all studies, increasing dietary BCAAs resulted in increased levels of circulating BCAAs. These effects, however, were heavily moderated by background dietary levels whereby on high BCAA diets, further increases were not reflected in the blood. Impaired glucose tolerance was associated with elevated dietary BCAAs, with the greatest effect occurring with a simultaneous increase in total protein intake. Effects of dietary BCAAs on plasma glucose, insulin, or HOMA emerged only when dietary macronutrient background was considered. We found that elevated dietary BCAAs increases % body fat, with largest increases in adiposity occurring when BCAAs are increased on a high protein, low carbohydrate dietary background. Finally, we found that increased dietary BCAAs were associated with increased food intake when the background diet was low in BCAAs. CONCLUSION: Our data highlights the interaction between BCAAs and background nutrition. We show that the effects of BCAAs on metabolic health cannot be studied in isolation but must be considered as part of complex mixture of dietary components.

Does diet influence aging? Evidence from animal studies.

Nutrition profoundly influences the risk for many age-related diseases. Whether nutrition influences human aging biology directly is less clear. Studies in different animal species indicate that reducing food intake ("caloric restriction" [CR]) can increase lifespan and delay the onset of diseases and the biological hallmarks of aging. Obesity has been described as "accelerated aging" and therefore the lifespan and health benefits generated by CR in both aging and obesity may occur via similar mechanisms. Beyond calorie intake, studies based on nutritional geometry have shown that protein intake and the interaction between dietary protein and carbohydrates influence age-related health and lifespan. Studies where animals are calorically restricted by providing free access to diluted diets have had less impact on lifespan than those studies where animals are given a reduced aliquot of food each day and are fasting between meals. This has drawn attention to the role of fasting in health and aging, and exploration of the health effects of various fasting regimes. Although definitive human clinical trials of nutrition and aging would need to be unfeasibly long and unrealistically controlled, there is good evidence from animal experiments that some nutritional interventions based on CR, manipulating dietary macronutrients, and fasting can influence aging biology and lifespan.

LC-N2G: a local consistency approach for nutrigenomics data analysis.

BACKGROUND: Nutrigenomics aims at understanding the interaction between nutrition and gene information. Due to the complex interactions of nutrients and genes, their relationship exhibits non-linearity. One of the most effective and efficient methods to explore their relationship is the nutritional geometry framework which fits a response surface for the gene expression over two prespecified nutrition variables. However, when the number of nutrients involved is large, it is challenging to find combinations of informative nutrients with respect to a certain gene and to test whether the relationship is stronger than chance. Methods for identifying informative combinations are essential to understanding the relationship between nutrients and genes. RESULTS: We introduce Local Consistency Nutrition to Graphics (LC-N2G), a novel approach for ranking and identifying combinations of nutrients with gene expression. In LC-N2G, we first propose a model-free quantity called Local Consistency statistic to measure whether there is non-random relationship between combinations of nutrients and gene expression measurements based on (1) the similarity between samples in the nutrient space and (2) their difference in gene expression. Then combinations with small LC are selected and a permutation test is performed to evaluate their significance. Finally, the response surfaces are generated for the subset of significant relationships. Evaluation on simulated data and real data shows the LC-N2G can accurately find combinations that are correlated with gene expression. CONCLUSION: The LC-N2G is practically powerful for identifying the informative nutrition variables correlated with gene expression. Therefore, LC-N2G is important in the area of nutrigenomics for understanding the relationship between nutrition and gene expression information.

Sex-specific metabolic responses to 6 hours of fasting during the active phase in young mice.

KEY POINTS: Night time/active phase food restriction for 6 h impaired glucose intolerance in young male and female mice. Females displayed increased capacity for lipogenesis and triglyceride storage in response to a short daily fast. Females had lower fasting insulin levels and an increased potential for utilizing fat for energy through ß-oxidation compared to males. The need for the inclusion of both sexes, and the treatment of sex as an independent variable, is emphasized within the context of this fasting regime. ABSTRACT: There is growing interest in understanding the mechanistic significance and benefits of fasting physiology in combating obesity. Increasing the fasting phase of a normal day can promote restoration and repair mechanisms that occur during the post-absorptive period. Most studies exploring the effect of restricting food access on mitigating obesity have done so with a large bias towards the use of male mice. Here, we disentangle the roles of sex, food intake and food withdrawal in the response to a short-term daily fasting intervention, in which food was removed for 6 h in the dark/active phase of young, 8-week-old mice. We showed that the removal of food during the dark phase impaired glucose tolerance in males and females, possibly due to the circadian disruption induced by this feeding protocol. Although both sexes demonstrated similar patterns of food intake, body composition and various metabolic markers, there were clear sex differences in the magnitude and extent of these responses. While females displayed enhanced capacity for lipogenesis and triglyceride storage, they also had low fasting insulin levels and an increased potential for utilizing available energy sources such as fat for energy through ß-oxidation. Our results highlight the intrinsic biological and metabolic disparities between male and female mice, emphasizing the growing need for the inclusion of both sexes in scientific research. Furthermore, our results illustrate sex-specific metabolic pathways that regulate lipogenesis, obesity and overall metabolic health.

Ingestion of resistant starch by mice markedly increases microbiome-derived metabolites.

Recent research has shown significant health benefits deriving from high-dietary fiber or microbiome-accessible carbohydrate consumption. Compared with native starch (NS), dietary resistant starch (RS) is a high microbiome-accessible carbohydrate that significantly alters the gut microbiome. The aim of this study was to determine the systemic metabolic effects of high microbiome-accessible carbohydrate. Male C57BL/6 mice were divided into 2 groups and fed either NS or RS for 18 wk (n = 20/group). Metabolomic analyses revealed that plasma levels of numerous metabolites were significantly different between the RS-fed and NS-fed mice, many of which are microbiome-derived. Most strikingly, we observed a 22-fold increase in gut microbiome-derived tryptophan metabolite indole-3-propionate (IPA), which was positively correlated with several gut microbiota, including Allobaculum, Bifidobacterium, and Lachnospiraceae, with Allobaculum having the most consistently increased abundance of all the IPA-associated taxa across all RS-fed mice. In addition, major changes were observed for metabolites solely or primarily metabolized in the gut (e.g., trimethylamine-N-oxide), metabolites that have a significant entero-hepatic circulation (i.e., bile acids), lipid metabolites (e.g., cholesterol sulfate), metabolites indicating increased energy turnover (e.g., tricarboxylic acid cycle intermediates and ketone bodies), and increased antioxidants such as reduced glutathione. Our findings reveal potentially novel mediators of high microbiome-accessible carbohydrate-derived health benefits.-Koay,Y. C., Wali. J. A., Luk, A. W. S., Macia, L., Cogger, V. C., Pulpitel, T. J., Wahl, D., Solon-Biet, S. M., Holmes, A., Simpson, S. J., O'Sullivan, J. F. Ingestion of resistant starch by mice markedly increases microbiome-derived metabolites.

Geometric framework reveals that a moderate protein, high carbohydrate intake is optimal for severe burn injury in mice.

Nutritional therapy is a cornerstone of burns management. The optimal macronutrient intake for wound healing after burn injury has not been identified, although high-energy, high-protein diets are favoured. The present study aimed to identify the optimal macronutrient intake for burn wound healing. The geometric framework (GF) was used to analyse wound healing after a 10 % total body surface area contact burn in mice ad libitum fed one of the eleven high-energy diets, varying in macronutrient composition with protein (P5-60 %), carbohydrate (C20-75 %) and fat (F20-75 %). In the GF study, the optimal ratio for wound healing was identified as a moderate-protein, high-carbohydrate diet with a protein:carbohydrate:fat (P:C:F) ratio of 1:4:2. High carbohydrate intake was associated with lower mortality, improved body weight and a beneficial pattern of body fat reserves. Protein intake was essential to prevent weight loss and mortality, but a protein intake target of about 7 kJ/d (about 15 % of energy intake) was identified, above which no further benefit was gained. High protein intake was associated with delayed wound healing and increased liver and spleen weight. As the GF study demonstrated that an initial very high protein intake prevented mortality, a very high-protein, moderate-carbohydrate diet (P40:C42:F18) was specifically designed. The dynamic diet study was also designed to combine and validate the benefits of an initial very high protein intake for mortality, and subsequent moderate protein, high carbohydrate intake for optimal wound healing. The dynamic feeding experiment showed switching from an initial very high-protein diet to the optimal moderate-protein, high-carbohydrate diet accelerated wound healing whilst preventing mortality and liver enlargement.

Aging, lifestyle and dementia.

Aging is the greatest risk factor for most diseases including cancer, cardiovascular disorders, and neurodegenerative disease. There is emerging evidence that interventions that improve metabolic health with aging may also be effective for brain health. The most robust interventions are non-pharmacological and include limiting calorie or protein intake, increasing aerobic exercise, or environmental enrichment. In humans, dietary patterns including the Mediterranean, Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) and Okinawan diets are associated with improved age-related health and may reduce neurodegenerative disease including dementia. Rapamycin, metformin and resveratrol act on nutrient sensing pathways that improve cardiometabolic health and decrease the risk for age-associated disease. There is some evidence that they may reduce the risk for dementia in rodents. There is a growing recognition that improving metabolic function may be an effective way to optimize brain health during aging.

Dietary macronutrient content, age-specific mortality and lifespan.

Protein and calorie restrictions extend median lifespan in many organisms. However, studies suggest that among-individual variation in the age at death is also affected. Ultimately, both of these outcomes must be caused by effects of nutrition on underlying patterns of age-specific mortality (ASM). Using model life tables, we tested for effects of dietary macronutrients on ASM in mice ( Mus musculus). High concentrations of protein and fat relative to carbohydrates were associated with low life expectancy and high variation in the age at death, a result caused predominantly by high mortality prior to middle age. A lifelong diet comprising the ratio of macronutrients self-selected by mouse (in early adulthood) was associated with low mortality up until middle age, but higher late-life mortality. This pattern results in reasonably high life expectancy, but very low variation in the age at death. Our analyses also indicate that it may be possible to minimize ASM across life by altering the ratio of dietary protein to carbohydrate in the approach to old age. Mortality in early and middle life was minimized at around one-part protein to two-parts carbohydrate, whereas in later life slightly greater than equal parts protein to carbohydrate reduced mortality.

The nutritional geometry of liver disease including non-alcoholic fatty liver disease.

Nutrition has a profound effect on chronic liver disease, especially non-alcoholic fatty liver disease (NAFLD). Most observational studies and clinical trials have focussed on the effects of total energy intake, or the intake of individual macronutrients and certain micronutrients, such as vitamin D, on liver disease. Although these studies have shown the importance of nutrition on hepatic outcomes, there is not yet any unifying framework for understanding the relationship between diet and liver disease. The Geometric Framework for Nutrition (GFN) is an innovative model for designing nutritional experiments or interpreting nutritional data that can determine the effects of nutrients and their interactions on animal behaviour and phenotypes. Recently the GFN has provided insights into the relationship between dietary energy and macronutrients on obesity and ageing in mammals including humans. Mouse studies using the GFN have disentangled the effects of macronutrients on fatty liver and the gut microbiome. The GFN is likely to play a significant role in disentangling the effects of nutrients on liver disease, especially NAFLD, in humans.

Macronutrient balance, reproductive function, and lifespan in aging mice.

In invertebrates, reproductive output and lifespan are profoundly impacted by dietary macronutrient balance, with these traits achieving their maxima on different diet compositions, giving the appearance of a resource-based tradeoff between reproduction and longevity. For the first time in a mammal, to our knowledge, we evaluate the effects of dietary protein (P), carbohydrate (C), fat (F), and energy (E) on lifespan and reproductive function in aging male and female mice. We show that, as in invertebrates, the balance of macronutrients has marked and largely opposing effects on reproductive and longevity outcomes. Mice were provided ad libitum access to one of 25 diets differing in P, C, F, and E content, with reproductive outcomes assessed at 15 months. An optimal balance of macronutrients exists for reproductive function, which, for most measures, differs from the diets that optimize lifespan, and this response differs with sex. Maximal longevity was achieved on diets containing a P:C ratio of 1:13 in males and 1:11 for females. Diets that optimized testes mass and epididymal sperm counts (indicators of gamete production) contained a higher P:C ratio (1:1) than those that maximized lifespan. In females, uterine mass (an indicator of estrogenic activity) was also greatest on high P:C diets (1:1) whereas ovarian follicle number was greatest on P:C 3:1 associated with high-F intakes. By contrast, estrous cycling was more likely in mice on lower P:C (1:8), and the number of corpora lutea, indicative of recent ovulations, was greatest on P:C similar to those supporting greatest longevity (1:11).

Nutritional geometry of paternal effects on embryo mortality.

Well-established causal links exist between maternal nutritional deficits and embryo health and viability. By contrast, environmental effects operating through the father that could influence embryo mortality have seldom been examined. Yet, ejaculates can require non-trivial resource allocation, and seminal plasma components are increasingly recognized to exert wide-ranging effects on females and offspring, so paternal dietary effects on the embryo should be expected. We test for effects of varying levels of protein (P), carbohydrate (C) and caloric load in adult male diet on embryo mortality in Drosophila melanogaster We demonstrate that macronutrient balance and caloric restriction exert significant effects, and that nutritional effects are more impactful when a prior mating has occurred. Once-mated males produced embryos with marginally elevated mortality under high-caloric densities and a 1 : 8 P : C ratio. In contrast, embryos produced by twice-mated males were significantly more likely to die under male caloric restriction, an outcome that may have resulted from shifts in ejaculate quality and/or epigenetic paternal effects. Body nutrient reserves were strongly and predictably altered by diet, and body condition, in turn, was negatively related to embryo mortality. Thus, sire nutritional history and resultant shifts in metabolic state predict embryo viability and post-fertilization fitness outcomes.

The impact of low-protein high-carbohydrate diets on aging and lifespan.

Most research on nutritional effects on aging has focussed on the impact of manipulating single dietary factors such as total calorie intake or each of the macronutrients individually. More recent studies using a nutritional geometric approach called the Geometric Framework have facilitated an understanding of how aging is influenced across a landscape of diets that vary orthogonally in macronutrient and total energy content. Such studies have been performed using ad libitum feeding regimes, thus taking into account compensatory feeding responses that are inevitable in a non-constrained environment. Geometric Framework studies on insects and mice have revealed that diets low in protein and high in carbohydrates generate longest lifespans in ad libitum-fed animals while low total energy intake (caloric restriction by dietary dilution) has minimal effect. These conclusions are supported indirectly by observational studies in humans and a heterogeneous group of other types of interventional studies in insects and rodents. Due to compensatory feeding for protein dilution, low-protein, high-carbohydrate diets are often associated with increased food intake and body fat, a phenomenon called protein leverage. This could potentially be mitigated by supplementing these diets with interventions that influence body weight through physical activity and ambient temperature.

Dietary macronutrients and the aging liver sinusoidal endothelial cell.

Fenestrations are pores within the liver sinusoidal endothelial cells (LSECs) that line the sinusoids of the highly vascularized liver. Fenestrations facilitate the transfer of substrates between blood and hepatocytes. With pseudocapillarization of the hepatic sinusoid in old age, there is a loss of fenestrations. LSECs are uniquely exposed to gut-derived dietary and microbial substrates delivered by the portal circulation to the liver. Here we studied the effect of 25 diets varying in content of macronutrients and energy on LSEC fenestrations using the Geometric Framework method in a large cohort of mice aged 15 mo. Macronutrient distribution rather than total food or energy intake was associated with changes in fenestrations. Porosity and frequency were inversely associated with dietary fat intake, while fenestration diameter was inversely associated with protein or carbohydrate intake. Fenestrations were also linked to diet-induced changes in gut microbiome, with increased fenestrations associated with higher abundance of Firmicutes and reduced abundance of Bacteroidetes Diet-induced changes in levels of several fatty acids (C16:0, C19:0, and C20:4) were also significantly inversely associated with fenestrations, suggesting a link between dietary fat and modulation of lipid rafts in the LSECs. Diet influences fenestrations and these data reflect both the key role of the LSECs in clearing gut-derived molecules from the vascular circulation and the impact these molecules have on LSEC morphology.

New Horizons: Dietary protein, ageing and the Okinawan ratio.

Nutrition has profound effects on ageing and lifespan. Caloric restriction is the major nutritional intervention that historically has been shown to influence lifespan and/or healthspan in many animal models. Studies have suggested that a reduction in protein intake can also increase lifespan, albeit not as dramatically as caloric restriction. More recent research based on nutritional geometry has attempted to define the effects of nutrition on ageing over a broad landscape of dietary macronutrients and energy content. Such studies in insects and mice indicate that animals with ad libitum access to low-protein, high-carbohydrate diets have longest lifespans. Remarkably, the optimum content and ratio of dietary protein to carbohydrates for ageing in experimental animals are almost identical to those in the traditional diets of the long-lived people on the island of Okinawa.

The effects of paternal dietary fat versus sugar on offspring body composition and anxiety-related behavior.

Increasing evidence suggests that the pre-conception parental environment has long-term consequences for offspring health and disease susceptibility. Though much of the work in this field concentrates on maternal influences, there is growing understanding that fathers also play a significant role in affecting offspring phenotypes. In this study, we investigate effects of altering the proportion of dietary fats and carbohydrates on paternal and offspring body composition and anxiety-related behavior in C57Bl/6-JArc mice. We show that in an isocaloric context, greater dietary fat increased body fat and reduced anxiety-like behavior of studs, whereas increased dietary sucrose had no significant effect. These dietary effects were not reflected in offspring traits, rather, we found sex-specific effects that differed between offspring body composition and behavioral traits. This finding is consistent with past paternal effect studies, where transgenerational effects have been shown to be more prominent in one sex over the other. Here, male offspring of fathers fed high-fat diets were heavier at 10 weeks of age due to increased lean body mass, whereas paternal diet had no significant effect on female offspring body fat or lean mass. In contrast, paternal dietary sugar appeared to have the strongest effects on male offspring behavior, with male offspring of high-sucrose fathers spending less time in the closed arms of the elevated plus maze. Both high-fat and high-sugar paternal diets were found to reduce anxiety-like behavior of female offspring, although this effect was only evident when offspring were fed a control diet. This study provides new understanding of the ways in which diet can shape the behavior of fathers and their offspring and contribute to the development of dietary guidelines to improve obesity and mental health conditions, such as anxiety.

Maternal macronutrient intake effects on offspring macronutrient targets and metabolism.

OBJECTIVE: Exposure in utero to maternal diet can program offspring health and susceptibility to disease. Using C57BL6/JArc mice, we investigated how maternal dietary protein to carbohydrate balance influences male and female offspring appetite and metabolic health. METHODS: Dams were placed on either a low-protein (LP) or high-protein (HP) diet. Male and female offspring were placed on a food choice experiment post weaning and were then constrained to either a standard diet or Western diet. Food intake, body weight, and composition were measured, and various metabolic tests were performed at different timepoints. RESULTS: Offspring from mothers fed HP diets selected a higher protein intake and had increased body weight in early life relative to offspring from LP diet-fed dams. As predicted by protein leverage theory, higher protein intake targets led to increased food intake when offspring were placed on no-choice diets, resulting in greater body weight and fat mass. The combination of an HP maternal diet and a Western diet further exacerbated this obesity phenotype and led to long-term consequences for body composition and metabolism. CONCLUSIONS: This work could help explain the association between elevated protein intake in humans during early life and increased risk of obesity in childhood and later life.

The Relationship between Dietary Macronutrient Composition and Telomere Length Among US Adults.

The role of dietary macronutrients and energy intake in the aging process has been well-established. However, previous research has mainly focused on the association between leukocyte telomere length (LTL) and individual macronutrients, while the effects of macronutrient composition on LTL remain unclear. This cross-sectional analysis involved 4130 US adults (44.8 ± 17.0 years; 51% female) from the National Health and Nutrition Examination Survey during 1999-2002. A single 24-h dietary recall is used to collect dietary data. The relationship between dietary macronutrient composition and LTL is examined using three-dimensional generalized additive models. After adjustment for age, sex, ethnicity, education, physical activity, BMI, and dietary quality, a three-dimensional association of macronutrient composition with LTL (P = 0.02) is revealed. Diets lower in protein (5-10%), higher in carbohydrates (75%), and lower in fat (15-20%) are associated with the longest LTL corresponding to 7.7 years of slower biological aging. Diets lowest in protein (5%) and carbohydrate (40%), while highest in dietary fat (55%) are associated with the shortest LTL, corresponding to accelerated biological aging of 4.4 years. The associations appeared magnified with higher energy intake. These findings support a complex relationship between dietary macronutrients and biological aging independent of diet quality.

Toward reconciling the roles of FGF21 in protein appetite, sweet preference, and energy expenditure.

Fibroblast growth factor 21 (FGF21), an endocrine signal robustly increased by protein restriction independently of an animal's energy status, exerts profound effects on feeding behavior and metabolism. Here, we demonstrate that considering the nutritional contexts within which FGF21 is elevated can help reconcile current controversies over its roles in mediating macronutrient preference, food intake, and energy expenditure. We show that FGF21 is primarily a driver of increased protein intake in mice and that the effect of FGF21 on sweet preference depends on the carbohydrate balance of the animal. Under no-choice feeding, FGF21 infusion either increased or decreased energy expenditure depending on whether the animal was fed a high- or low-energy diet, respectively. We show that while the role of FGF21 in mediating feeding behavior is complex, its role in promoting protein appetite is robust and that the effects on sweet preference and energy expenditure are macronutrient-state-dependent effects of FGF21.

An integrative approach to dietary balance across the life course.

Animals require specific blends of nutrients that vary across the life course and with circumstances, e.g., health and activity levels. Underpinning and complicating these requirements is that individual traits may be optimized on different dietary compositions leading to nutrition-mediated trade-offs among outcomes. Additionally, the food environment may constrain which nutrient mixtures are achievable. Natural selection has equipped animals for solving such multi-dimensional, dynamic challenges of nutrition, but little is understood about the details and their theoretical and practical implications. We present an integrative framework, nutritional geometry, which models complex nutritional interactions in the context of multiple nutrients and across levels of biological organization (e.g., cellular, individual, and population) and levels of analysis (e.g., mechanistic, developmental, ecological, and evolutionary). The framework is generalizable across different situations and taxa. We illustrate this using examples spanning insects to primates and settings (laboratory, and the wild), and demonstrate its relevance for human health.