Camera collars reveal macronutrient balancing in free-ranging male moose during summer.

Understanding how the nutritional properties of food resources drive foraging choices is important for the management and conservation of wildlife populations. For moose (Alces alces), recent experimental and observational studies during the winter have shown macronutrient balancing between available protein (AP) and highly metabolizable macronutrients (total non-structural carbohydrates [TNC] and lipids). Here, we combined the use of continuous-recording camera collars with plant nutrient analyses and forage availability measurements to obtain a detailed insight into the food and nutritional choices of three wild moose in Norway over a 5-day period in summer. We found that moose derived their macronutrient energy primarily from carbohydrates (74.2%), followed by protein (13.1%), and lipids (12.7%). Diets were dominated by deciduous tree browse (71%). Willows (Salix spp.) were selected for and constituted 51% of the average diet. Moose consumed 25 different food items during the study period of which 9 comprised 95% of the diet. Moose tightly regulated their intake of protein to highly metabolizable macronutrients (AP:TNC + lipids) to a ratio of 1:2.7 (0.37 ± 0.002SD). They did this by feeding on foods that most closely matched the target macronutrient ratio such as Salix spp., or by combining nutritionally imbalanced foods (complementary feeding) in a non-random manner that minimized deviations from the intake target. The observed patterns of macronutrient balancing aligned well with the findings of winter studies. Differential feeding on nutritionally balanced downy birch (Betula pubescens) leaves versus imbalanced twigs+leaves across moose individuals indicated that macronutrient balancing may occur on as fine a scale as foraging bites on a single plant species. Utilized forages generally met the suggested requirement thresholds for the minerals calcium, phosphorus, copper, molybdenum, and magnesium but tended to be low in sodium. Our findings offer new insights into the foraging behavior of a model species in ungulate nutritional ecology and contribute to informed decision-making in wildlife and forest management.

On the pathogenesis of obesity: causal models and missing pieces of the puzzle.

Application of the physical laws of energy and mass conservation at the whole-body level is not necessarily informative about causal mechanisms of weight gain and the development of obesity. The energy balance model (EBM) and the carbohydrate-insulin model (CIM) are two plausible theories, among several others, attempting to explain why obesity develops within an overall common physiological framework of regulation of human energy metabolism. These models have been used to explain the pathogenesis of obesity in individuals as well as the dramatic increases in the prevalence of obesity worldwide over the past half century. Here, we summarize outcomes of a recent workshop in Copenhagen that brought together obesity experts from around the world to discuss causal models of obesity pathogenesis. These discussions helped to operationally define commonly used terms; delineate the structure of each model, particularly focussing on areas of overlap and divergence; challenge ideas about the importance of purported causal factors for weight gain; and brainstorm on the key scientific questions that need to be answered. We hope that more experimental research in nutrition and other related fields, and more testing of the models and their predictions will pave the way and provide more answers about the pathogenesis of obesity than those currently available.

Diet modifies the association between alcohol consumption and severe alcohol-related liver disease incidence.

It is elusive why some heavy drinkers progress to severe alcohol-related liver disease (ALD) while others do not. This study aimed to investigate if the association between alcohol consumption and severe ALD is modified by diet. This prospective study included 303,269 UK Biobank participants. Alcohol consumption and diet were self-reported. The diet score was created from 4 items selected using LASSO. Cox proportional hazard model showed that the diet score was monotonically associated with severe ALD risk, adjusted for sociodemographics, lifestyle factors, and alcohol consumption. Relative excess risk due to interaction analysis indicated that having a higher ALD diet score and a higher alcohol consumption simultaneously confers to 2.44 times (95% CI: 1.06-3.83) higher risk than the sum of excess risk of each factor. In this work, we show that people who have a poor diet might be more susceptible to severe ALD due to alcohol consumption.

Increased intake of tree forage by moose is associated with intake of crops rich in nonstructural carbohydrates.

Animals representing a wide range of taxonomic groups are known to select specific food combinations to achieve a nutritionally balanced diet. The nutrient balancing hypothesis suggests that, when given the opportunity, animals select foods to achieve a particular target nutrient balance, and that balancing occurs between meals and between days. For wild ruminants who inhabit landscapes dominated by human land use, nutritionally imbalanced diets can result from ingesting agricultural crops rich in starch and sugar (nonstructural carbohydrates [NCs]), which can be provided to them by people as supplementary feeds. Here, we test the nutrient balancing hypothesis by assessing potential effects that the ingestion of such crops by Alces alces (moose) may have on forage intake. We predicted that moose compensate for an imbalanced intake of excess NC by selecting tree forage with macro-nutritional content better suited for their rumen microbiome during wintertime. We applied DNA metabarcoding to identify plants in fecal and rumen content from the same moose during winter in Sweden. We found that the concentration of NC-rich crops in feces predicted the presence of Picea abies (Norway spruce) in rumen samples. The finding is consistent with the prediction that moose use tree forage as a nutritionally complementary resource to balance their intake of NC-rich foods, and that they ingested P. abies in particular (normally a forage rarely eaten by moose) because it was the most readily available tree. Our finding sheds new light on the foraging behavior of a model species in herbivore ecology, and on how habitat alterations by humans may change the behavior of wildlife.

Rapid benefits in older age from transition to whole food diet regardless of protein source or fat to carbohydrate ratio: Arandomised control trial.

Plant-based diets reduces the risk of chronic conditions. The interaction between protein source and other macronutrients-fat (F) and carbohydrate (C)-has yet to be investigated. The aim was to assess the main and interactive effects of protein-source (plant vs. animal) and F:C (high or low) and the transition from an Australian diet to a whole food diet on various health markers in older individuals. This single-blinded, parallel, randomised experimental trial used a 2 × 2 factorial design to compare pro-vegetarian (70:30 plant to animal) versus omnivorous (50:50 plant to animal) diets at 14% protein and varying fat-to-carbohydrate ratios (high fat ~40% vs. low fat ~30%) over 4 weeks. Study foods were provided, alcohol consumption was discouraged, and dietary intake was determined through food records. Analysis included both RCT and observational data. Changes in appetite, palatability of diets, and dietary intake were assessed. Body composition, muscle strength, function, gut microbiome, and cardiometabolic health parameters were measured. Data from 113 (of the 128 randomised) individuals aged 65-75 years were analysed. Pro-vegetarian diets reduced diastolic blood pressure, total cholesterol and glucose levels. Moreover, the overall sample exhibited increased short-chain fatty acids and FGF21 levels, as well as improvements in body composition, function, and cardio-metabolic parameters irrespective of dietary treatment. Transitioning to a diet rich in fruit, vegetables, fibre, and moderate protein was associated with improved health markers in older age, with added benefits from pro-vegetarian diets. Further research on long-term effects is needed.

'Dust you shall eat': The complex nutritional and functional considerations underlying a simple diet.

Animals assimilate macronutrients and mineral nutrients in specific quantities and ratios to maximise fitness. To achieve this, animals must ingest different foods that contain the needed nutrients or facilitate the digestion of those nutrients. We explored how these multidimensional considerations affect the desert isopods (Hemilepistus reaumuri) curious food selection, using field and laboratory experiments. Wild isopods consumed three-fold more macronutrient-poor biological soil crust (BSC) than plant litter. Isopods tightly regulated macronutrient and calcium intake, but not phosphorus when eating the two natural foods and when artificial calcium and phosphorus sources substituted the BSC. Despite the equivalent calcium ingestion, isopods performed better when eating BSC compared to artificial foods. Isopods that consumed BSC sterilised by gamma-radiation ate more but grew slower than isopods that ate live BSC, implying that ingested microorganisms facilitate litter digestion. Our work highlights the need to reveal the multifaceted considerations that affect food-selection when exploring trophic-interactions.

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.

Macronutrient composition of plant-based diets and breast cancer risk: the E3N prospective cohort study.

PURPOSE: Recent evidence suggests that plant-based diets may reduce the risk of breast cancer (BC). However, the macronutrient composition of plant-based diets and its potential impact on BC risk has not been well explored. This analysis investigated the association of macronutrient composition with BC risk across a spectrum of plant-based diet indexes using a multidimensional approach. DESIGN: This study followed 64,655 participants from the Etude Epidémiologique auprès de femmes de la Mutuelle Générale de l'Education Nationale (E3N) cohort from 1993 to 2014. Diets were evaluated using validated 208-item diet history questionnaires at baseline (1993) and follow-up (2005), to calculate adherence to the overall plant-based diet (PDI), healthful plant-based diet (hPDI), and unhealthful plant-based diet (uPDI). The association of macronutrient composition with BC risk was assessed via generalized additive time-dependent Cox models across different levels of these indexes. Response surfaces were generated to visualize compositional associations at the 25th, 50th, and 75th percentile of each index (low, moderate, and high). RESULTS: A total of 3,932 incident BC cases were identified during the 21-year follow-up. There was a significant association between macronutrient composition and BC risk for hPDI, uPDI, and PDI (all P < 0.001). Akaike information criterion favored the hPDI model for characterizing the association between macronutrients and BC. BC risk was highest for individuals with a lower hPDI score who also consumed a diet containing lower protein (10%), lower carbohydrate (35%), and higher fat (55%). The lowest risk of BC was observed in those with higher hPDI scores with the lowest intake of protein (10%). At higher PDI and uPDI, diets containing higher protein (30%) and fat (45%) had the highest BC risk. CONCLUSION: These results demonstrate a complex relationship between macronutrient composition, plant-based diet quality, and BC risk. Further research is needed to examine specific foods that may be driving these associations. REGISTRY: The protocol is registered at clinicaltrials.gov as NCT03285230.

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.

Identification of a Novel Peptide with Alcohol Dehydrogenase Activating Ability from Ethanol-Induced Lactococcus lactis: A Combined In Silico Prediction and In Vivo Validation.

Alcohol dehydrogenase (ADH) is a crucial rate-limiting enzyme in alcohol metabolism. Our previous research found that ethanol-induced intracellular extracts of Lactococcus lactis (L. lactis) could enhance alcohol metabolism in mice, but the responsible compounds remain unidentified. The study aimed to screen potential ADH-activating peptides from ethanol-induced L. lactis using virtual screening and molecular docking calculation. Among them, the pentapeptide FAPEG might bind to ADH through hydrophobic interaction and hydrogen bonds, then enhancing ADH activity. Spectroscopy analysis further investigated the peptide-enzyme interaction between FAPEG and ADH, including changes in the amino acid residue microenvironment and secondary structural alterations. Furthermore, FAPEG could protect against alcoholic liver injury (ALI) in mice by reducing blood alcohol concentration, enhancing the activity of antioxidant and alcohol metabolism enzymes, and attenuating alcohol-induced hepatotoxicity, which was related to the activation of the Nrf2/keap1/HO-1 signaling pathway. The study provided preliminary evidence that the generation of ADH-activating peptides in ethanol-induced L. lactis has the potential in preventing ALI in mice using in silico prediction and in vivo validation approaches.

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.

Evidence for protein leverage on total energy intake, but not body mass index, in a large cohort of older adults.

BACKGROUND: Protein leverage (PL) is the phenomenon of consuming food until absolute intake of protein approaches a 'target value', such that total energy intake (TEI) varies passively with the ratio of protein: non-protein energy (fat + carbohydrate) in the diet. The PL hypothesis (PLH) suggests that the dilution of protein in energy-dense foods, particularly those rich in carbohydrates and fats, combines with protein leverage to contribute to the global obesity epidemic. Evidence for PL has been reported in younger adults, children and adolescents. This study aimed to test for PL and the protein leverage hypothesis (PLH) in a cohort of older adults. METHODS: We conducted a retrospective analysis of dietary intake in a cohort of 1699 community-dwelling older adults aged 67-84 years from the NuAge cohort. We computed TEI and the energy contribution (EC) from each macronutrient. The strength of leverage of macronutrients was assessed through power functions ( TEI = µ * EC L ). Body mass index (BMI) was calculated, and mixture models were fitted to predict TEI and BMI from macronutrients' ECs. RESULTS: In this cohort of older adults, 53% of individuals had obesity and 1.5% had severe cases. The mean TEI was 7673 kJ and macronutrients' ECs were 50.4%, 33.2% and 16.4%, respectively for carbohydrates, fat, and protein. There was a strong negative association (L = -0.37; p < 0.001) between the protein EC and TEI. Each percent of energy intake from protein reduced TEI by 77 kJ on average, ceteris paribus. However, BMI was unassociated with TEI in this cohort. CONCLUSIONS: Findings indicate clear evidence for PL on TEI, but not on BMI, likely because of aging, body composition, sarcopenia, or protein wasting.

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.

In Situ Provisioning Wildlife with Food, Water, or Shelter after Bushfires: Using a One Welfare Framework to Guide Responses.

Australia's 2019-2020 bushfires had a devastating impact on animals, humans, and ecosystems. They also demonstrated the lack of evidence or guidance for wildlife provisioning in response to severe fire events when volunteers and wildlife organisations rose to respond. In addition, the unprecedented scale and intensity of the fires and an absence of institutional support for wildlife provisioning meant that well-intentioned interventions were largely uncoordinated and lacked clear short-term, mid-term, and long-term objectives. Fundamentally, a lack of consensus was revealed on whether any such interventions are advisable. Given the strong evidence indicating that future bushfire seasons will become longer and more intense in Australia and elsewhere, the welfare and survival of millions of wild animals are at risk every year. Understanding the impacts of supplementary resource interventions and contributing to the development of best practice information is crucial to inform the response to the next major fire event. Here, we contextualize the arguments for and against provisioning within a 'One Welfare' framework that recognizes that animal welfare, biodiversity, and the environment are intertwined with human welfare and community resilience. We propose that the One Welfare approach can facilitate appropriate consideration of the extant scientific and lay literature; local legislation; views of stakeholders; emerging data; and modelling from historic fire events. As a further step, we see merit in engaging with wildlife provisioners and the broader conservation community to build an evidence base for future wildlife provisioning activities. From an informed position, we can encourage beneficial interventions and reduce the risk of negative outcomes. Finally, we propose controlled experiments (e.g., using hazard reduction burns), ongoing data collection using emergent technology, and longitudinal analysis to address shifting research priorities as the climate changes. We conclude that the ordered collection of the necessary evidence relevant to each of the three stakeholder groups in the One Welfare framework has the greatest potential to support an informed policy platform on wildlife provisioning across Australia that is feasible, legal, and sustainable.

Systems map of interventions to improve dietary intake of pre-school aged children: A scoping review.

Implementation and sustaining impact of early childhood nutrition interventions in practice remains a challenge. An understanding of the extent to which determinants across multiple levels of the food system are being addressed may improve success. This literature review aimed to synthesise the evidence on interventions targeting dietary intake and eating behaviours in preschool children using a systems approach. Eligible studies included intervention studies targeting the dietary intake of preschool children aged 2-5 years in high income countries, published in English after January 2000. Interventions were categorised to the Determinants of Nutrition and Eating (DONE) framework for children developed and evaluated by experts across multiple fields. The framework maps and ranks 411 factors driving eating behaviours and nutrition and can be used to systematically summarise determinants. DONE ranks each determinant for its perceived research priority. A total of 160 eligible studies were identified. Most interventions targeted interpersonal (n = 101, 63.1%) and individual (n = 85, 53.1%) level determinants, with fewer targeting environmental (n = 55, 34.4%) and policy level (n = 17, 10.6%) determinants. The most frequently addressed determinants were Parental Resources and Risk Factors (n = 85) and Children's Food Knowledge, Skills and Abilities (n = 67). These determinants had a Moderate research priority rating. Home Food Availability and Accessibility at the environmental level is classified as the highest research priority, however, only 15 of 160 interventions addressed this determinant. This review highlights home food availability and accessibility as potential leverage points for future interventions to improve children's dietary intake and eating behaviours.

Towards an integrated understanding of dietary phenotypes.

Diet and nutrition comprise a complex, multi-faceted interface between animal biology and food environments. With accumulating information on the many facets of this association arises a need for systems-based approaches that integrate dietary components and their links with ecology, feeding, post-ingestive processes and the functional and ecological consequences of these interactions. We briefly show how a modelling approach, nutritional geometry, has used the experimental control afforded in laboratory studies to begin to unravel these links. Laboratory studies, however, have limited ability to establish whether and how the feeding and physiological mechanisms interface with realistic ecological environments. We next provide an overview of observational field studies of free-ranging primates that have examined this, producing largely correlative data suggesting that similar feeding mechanisms operate in the wild as in the laboratory. Significant challenges remain, however, in establishing causal links between feeding, resource variation and physiological processes in the wild. We end with a more detailed account of two studies of temperate primates that have capitalized on the discrete variation provided by seasonal environments to strengthen causal inference in field studies and link patterns of intake to dynamics of nutrient processing. This article is part of the theme issue 'Food processing and nutritional assimilation in animals'.

Nutritional, environmental and economic impacts of ultra-processed food consumption in Australia.

OBJECTIVE: To quantify the full life cycle impacts of ultra-processed foods (UPF) for key environmental, economic and nutritional indicators to identify trade-offs between UPF contribution to broad-scope sustainability. DESIGN: Using 24-h dietary recalls along with an input-output database for the Australian economy, dietary environmental and economic impacts were quantified in this national representative cross-sectional analysis. Food items were classified into non-UPF and UPF using the NOVA system, and dietary energy contribution from non-UPF and UPF fractions in diets was estimated. Thereafter, associations between nutritional, environmental and economic impacts of non-UPF and UPF fractions of diets were examined using a multi-dimensional nutritional geometry representation. SETTING: National Nutrition and Physical Activity Survey 2011-2012 of Australia. PARTICIPANTS: Respondents (n 5344) aged > 18 years with 1 d of 24-h dietary recall data excluding respondents with missing values and outlier data points and under reporters. RESULTS: Australian diets rich in UPF were associated with reduced nutritional quality, high greenhouse gas emissions, energy use, and increased employment and income associated with the food supply chains. The environmental and economic impacts associated with the UPF portion of diets become more distinct when the diets are standardised to average protein recommendation. CONCLUSION: Increased consumption of UPF has socio-economic benefits, but this comes with adverse effects on the environment and public health. Consideration of such trade-offs is important in identifying policy and other mechanisms regarding UPF for establishing healthy and sustainable food systems.

Evidence for the protein leverage hypothesis in preschool children prone to obesity.

BACKGROUND & AIMS: The protein leverage hypothesis (PLH) proposed that strict regulation of protein intake drives energy overconsumption and obesity when diets are diluted by fat and/or carbohydrates. Evidence about the PLH has been found in adults, while studies in children are limited. Thus, we aimed to test the PLH by assessing the role of dietary protein on macronutrients, energy intake, and obesity risk using data from preschool children followed for 1.3 years. METHODS: 553 preschool children aged 2-6 years from the 'Healthy Start' project were included. EXPOSURES: The proportion of energy intake from protein, fat, and carbohydrates collected from a 4-day dietary record. OUTCOMES: Energy intake, BMI z-score, fat mass (FM) %, waist- (WHtR) and hip-height ratio (HHtR). Power function analysis was used to test the leverage of protein on energy intake. Mixture models were used to explore interactive associations of macronutrient composition on all these outcomes, with results visualized as response surfaces on the nutritional geometry. RESULTS: Evidence for the PLH was confirmed in preschool children. The distribution of protein intake (% of MJ, IQR: 3.2) varied substantially less than for carbohydrate (IQR: 5.7) or fat (IQR: 6.3) intakes, suggesting protein intake is most tightly regulated. Absolute energy intake varied inversely with dietary percentage energy from protein (L = -0.14, 95% CI: -0.25, -0.04). Compared to children with high fat or carbohydrate intakes, children with high dietary protein intake (>20% of MJ) had a greater decrease in WHtR and HHtR over the 1.3-year follow-up, offering evidence for the PLH in prospective analysis. But no association was observed between macronutrient distribution and changes in BMI z-score or FM%. CONCLUSIONS: In this study in preschool children, protein intake was the most tightly regulated macronutrient, and energy intake was an inverse function of dietary protein concentration, indicating the evidence for protein leverage. Increases in WHtR and HHtR were principally associated with the dietary protein dilution, supporting the PLH. These findings highlight the importance of protein in children's diets, which seems to have significant implications for childhood obesity risk and overall health.