Protein Leverage Research Articles

Macronutrient interactions and models of obesity: Insights from nutritional geometry.

The global obesity epidemic results from a complex interplay of genetic and environmental factors, with diet being a prominent modifiable element driving weight gain and adiposity. Although excess intake of energetic macronutrients is implicated in causing obesity, ongoing debate centers on whether sugar or fat or both are driving the rising obesity rates. This has led to competing models of obesity such as the "Carbohydrate Insulin Model", the "Energy Balance Model", and the "Fructose Survival Hypothesis". Conflicting evidence from studies designed to focus on individual energetic macronutrients or energy rather than macronutrient mixtures underlies this disagreement. Recent research in humans and animals employing the nutritional geometry framework (NGF) emphasizes the importance of considering interactions among dietary components. Protein interacts with carbohydrates, fats, and dietary energy density to influence both calorie intake ("protein leverage") and, directly and indirectly, metabolic physiology and adiposity. Consideration of these interactions can help to reconcile different models of obesity, and potentially cast new light on obesity interventions.

Macronutrients in Human Milk and Early Childhood Growth-Is Protein the Main Driver?

Infant growth trajectories reflect current health status and may predict future obesity and metabolic diseases. Human milk is tailored to support optimal infant growth. However, nutrient intake rather than milk composition more accurately predicts growth outcomes. Although the role of protein leverage in infant growth is unclear, protein intake is important for early infancy growth. This study of exclusively breastfeeding mothers with allergies (n = 161) from the Infant Fish Oil Supplementation Study assessed relationships between intake of human milk macronutrients and infant growth. Human milk fat, protein and lactose concentrations were measured at 3 months postpartum, and infant daily intakes were estimated using an average milk intake of 800 mL/day. Higher human milk protein:energy ratio was associated with higher weight-for-age z-score at 2.5 years compared to 3 months and higher body mass index-for-age z-score change (6 months to 1 year compared to 3-6 months). Maternal atopy and birth season (summer) were negatively associated with human milk lactose concentration. Passive smoke exposure was associated with reduced energy and fat concentrations and increased lactose:energy ratio. Our results indicate that intake of human milk macronutrients may impact early childhood growth.

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

BackgroundA 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). ObjectivesWe 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. MethodsFood 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. ResultsPL 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. ConclusionsMice 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.

Maternal macronutrient intake effects on offspring macronutrient targets and metabolism.

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. 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. 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. 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.

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

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. 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 ( ). Body mass index (BMI) was calculated, and mixture models were fitted to predict TEI and BMI from macronutrients' ECs. 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. Findings indicate clear evidence for PL on TEI, but not on BMI, likely because of aging, body composition, sarcopenia, or protein wasting.

The fructose survival hypothesis as a mechanism for unifying the various obesity hypotheses.

The pathogenesis of obesity remains contested. Although genetics is important, the rapid rise in obesity with Western culture and diet suggests an environmental component. Today, some of the major hypotheses for obesity include the energy balance hypothesis, the carbohydrate-insulin model, the protein-leverage hypothesis, and the seed oil hypothesis. Each hypothesis has its own support, creating controversy over their respective roles in driving obesity. Here we propose that all hypotheses are largely correct and can be unified by another dietary hypothesis, the fructose survival hypothesis. Fructose is unique in resetting ATP levels to a lower level in the cell as a consequence of suppressing mitochondrial function, while blocking the replacement of ATP from fat. The low intracellular ATP levels result in carbohydrate-dependent hunger, impaired satiety (leptin resistance), and metabolic effects that result in the increased intake of energy-dense fats. This hypothesis emphasizes the unique role of carbohydrates in stimulating intake while fat provides the main source of energy. Thus, obesity is a disorder of energy metabolism, in which there is low usable energy (ATP) in the setting of elevated total energy. This leads to metabolic effects independent of excess energy while the excess energy drives weight gain.

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

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. 553 preschool children aged 2-6 years from the 'Healthy Start' project were included. The proportion of energy intake from protein, fat, and carbohydrates collected from a 4-day dietary record. 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. 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%. 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.

Protein appetite as an integrator in the obesity system: the protein leverage hypothesis.

Despite the large volume and extensive range of obesity research, there is substantial disagreement on the causes and effective preventative strategies. We suggest the field will benefit from greater emphasis on integrative approaches that examine how various potential contributors interact, rather than regarding them as competing explanations. We demonstrate the application of nutritional geometry, a multi-nutrient integrative framework developed in the ecological sciences, to obesity research. Such studies have shown that humans, like many other species, regulate protein intake more strongly than other dietary components, and consequently if dietary protein is diluted there is a compensatory increase in food intake-a process called protein leverage. The protein leverage hypothesis (PLH) proposes that the dilution of protein in modern food supplies by fat and carbohydrate-rich highly processed foods has resulted in increased energy intake through protein leverage. We present evidence for the PLH from a variety of sources (mechanistic, experimental and observational), and show that this mechanism is compatible with many other findings and theories in obesity research. This article is part of a discussion meeting issue 'Causes of obesity: theories, conjectures and evidence (Part II)'.

Associations between protein to non-protein ratio and intakes of other dietary components in a cohort aged 65-75 years: the Nutrition for Healthy Living Study.

Diets with a low proportion of energy from protein have shown to cause overconsumption of non-protein energy, known as Protein Leverage. Older adults are susceptible to nutritional inadequacy. The aim was to investigate associations between protein to non-protein ratio (P:NP) and intakes of dietary components and assess the nutritional adequacy of individuals aged 65-75 years from the Nutrition for Healthy Living (NHL) Study. Cross-sectional. Nutritional intakes from seven-day weighed food records were compared with the Nutrient Reference Values for Australia and New Zealand, Australian Guide to Healthy Eating, Australian Dietary Guidelines and World Health Organisation Free Sugar Guidelines. Associations between P:NP and intakes of dietary components were assessed through linear regression analyses. NHL Study. 113 participants. Eighty-eight (59 female and 29 male) with plausible dietary data had a median (interquartile range) age of 69 years (67-71), high education level (86 %) and sources of income apart from the age pension (81 %). Substantial proportions had intakes below recommendations for dairy and alternatives (89 %), wholegrain (89 %) and simultaneously exceeded recommendations for discretionary foods (100 %) and saturated fat (92 %). In adjusted analyses, P:NP (per 1 % increment) was associated with lower intakes of energy, saturated fat, free sugar and discretionary foods and higher intakes of vitamin B12, Zn, meat and alternatives, red meat, poultry and wholegrain % (all P < 0·05). Higher P:NP was associated with lower intakes of energy, saturated fat, free sugar and discretionary. Our study revealed substantial nutritional inadequacy in this group of higher socio-economic individuals aged 65-75 years.

Testing for Protein Leverage in Patients with Gastric Bypass: A Pilot Study

Introduction: Protein leverage (PL) is the phenomenon whereby a dominant appetite for protein drives overconsumption of energy with a decline in the ratio of protein to fat and carbohydrate in the diet. PL has been independently verified in several randomized control trials, and its predictions are supported by diet surveillance data. Our aim in the present study was to test whether surgical intervention through gastric bypass will ameliorate the PL effect. Methods: Ten patients with gastric bypass (2–5 years postsurgical time) were given ad libitum access to study food comprising 10%, 15%, or 25% protein and no access to other foods for 3 days while controlling food palatability and variety. Food intake was measured, and energy and nutrient intakes were calculated. Body weight, blood chemistry, lipid profile, hormones (insulin, leptin, and ghrelin), and creatinine were determined before and after each experimental period. Results: The gastric bypass patients in our study did not show evidence for protein intake regulation as predicted under PL but ate to constant total energy intake on the 10%, 15%, and 25% protein diets with protein intake varying significantly. Patients lost weight in the three study periods, but significant weight loss was observed only on the 15% protein diet. Conclusion: Our results suggest that gastric bypass might disengage the PL mechanism, thus ameliorating an appetite-specific mechanism that drives energy overconsumption in modern food environments.

No strong evidence of the protein leverage hypothesis in pregnant women with obesity and their infants.

The goal of this study was to investigate the role of dietary protein on macronutrient and energy intake, maternal adiposity during pregnancy, and infant adiposity at birth. In 41 women with obesity, early-pregnancy (13-16 weeks) protein intake was assessed with food photography and expressed as a ratio of Estimated Average Requirements (EAR) in pregnancy for protein (0.88 g/kg/d), herein "protein balance." Energy intake was measured by the intake-balance method, gestational weight gain as grams per week, and fat mass by a three-compartment model. Spearman correlations and linear models were computed using R version 4.1.1 (p < 0.05 considered significant). Women had a mean (SD) age of 27.5 (4.8) years and a pregravid BMI of 34.4 (2.9), kg/m2 , and the majority were non-White (n = 23, 56.1%). Protein balance in early pregnancy was not significantly associated with energy intake across mid and mid/late pregnancy (β = 328.7, p = 0.30 and β = 286.2, p = 0.26, respectively) or gestational weight gain (β = 117.0, p = 0.41). Protein balance was inversely associated with fat mass in early, mid, and late pregnancy (β = -10.6, p = 0.01, β = -10.4, p = 0.03, β = -10.3, p = 0.03, respectively). Protein balance did not predict infant adiposity at birth (p > 0.05). Low protein intake may have been present before pregnancy, explaining early relationships with adiposity in this cohort. The protein leverage hypothesis is likely not implicated in the intergenerational transmission of obesity.

Evidence for protein leverage in a general population sample of children and adolescents

Background/ObjectivesThe strong regulation of protein intake can lead to overconsumption of total energy on diets with a low proportion of energy from protein, a process referred to as protein leverage. The protein leverage hypothesis posits that protein leverage explains variation in energy intake and potentially obesity in ecological settings. Here, we tested for protein leverage and the protein leverage hypothesis in children and adolescents.Subjects/MethodsA population sample of children, mean (SD) age 7.6 (0.4) years (n = 422), followed up at age 9.8 (0.4) years (n = 387) and at age 15.8 (0.4) years (n = 229), participating for the Physical Activity and Nutrition in Children (PANIC) study. Exposures: 4-day food records-related proportional energy intake of proteins, fats, and carbohydrates. Outcomes: energy intake, body mass index (BMI) z-score and dual-energy X-ray absorptiometry-related energy expenditure.ResultsProportional energy intake of proteins was inversely associated with energy intake following power functions at all 3 ages (mean [95%CI] strength of leverage of L = −0.36 [−0.47 to −0.25]; L = −0.26 [−0.37 to −0.15]; L = −0.25 [−0.38 to −0.13]; all P < 0.001). Mixture analysis indicated that variance in energy intake was associated primarily with the proportional intake of energy from proteins, not with either fats or carbohydrates. At all 3 ages, energy intake was not associated with BMI z-score but positively associated with energy expenditure (all P < 0.001).ConclusionsThis study provides evidence consistent with protein leverage in a population sample of children and adolescents. Increased energy intake on diets with lower protein content was counterbalanced by increased energy expenditure and therefore did not translate into increased adiposity.

Macronutrient (im)balance drives energy intake in an obesogenic food environment: An ecological analysis.

The protein leverage hypothesis (PLH) postulates that strong regulation of protein intake drives energy overconsumption and obesity when human diets are diluted by fat and carbohydrates. The two predictions of the PLH are that humans (i) regulate intake to maintain protein within a narrow range and that (ii) energy intake is an inverse function of percentage energy from protein because absolute protein intake is maintained within narrow limits. Multidimensional nutritional geometry was used to test the predictions of the PLH using dietary data from the Australian National Nutrition and Physical Activity Survey. Both predictions of the PLH were confirmed in a population setting: the mean protein intake was 18.4%, and energy intake decreased with increasing energy from protein (L=-0.18, p &lt; 0.0001). It was demonstrated that highly processed discretionary foods are a significant diluent of protein and associated with increased energy but not increased protein intake. These results support an integrated ecological and mechanistic explanation for obesity, in which low-protein highly processed foods lead to higher energy intake because of the biological response to macronutrient imbalance driven by a dominant appetite for protein. This study supports a central role for protein in the obesity epidemic, with significant implications for global health.

Weight gain during the menopause transition: Evidence for a mechanism dependent on protein leverage.

Weight gain during the menopause transition: Evidence for a mechanism dependent on protein leverage.

A Comparison of the Australian Dietary Guidelines to the NOVA Classification System in Classifying Foods to Predict Energy Intakes and Body Mass Index.

NOVA classification distinguishes foods by level of processing, with evidence suggesting that a high intake of ultra-processed foods (UPFs, NOVA category 4) leads to obesity. The Australian Dietary Guidelines, in contrast, discourage excess consumption of “discretionary foods” (DFs), defined according to their composition. Here, we (i) compare the classification of Australian foods under the two systems, (ii) evaluate their performance in predicting energy intakes and body mass index (BMI) in free-living Australians, and (iii) relate these outcomes to the protein leverage hypothesis of obesity. Secondary analysis of the Australian National Nutrition and Physical Activity Survey was conducted. Non-protein energy intake increased by 2.1 MJ (p < 0.001) between lowest and highest tertiles of DF intake, which was significantly higher than UPF (0.6 MJ, p < 0.001). This demonstrates that, for Australia, the DF classification better distinguishes foods associated with high energy intakes than does the NOVA system. BMI was positively associated with both DFs (−1. 0, p = 0.0001) and UPFs (−1.1, p = 0.0001) consumption, with no difference in strength of association. For both classifications, macronutrient and energy intakes conformed closely to the predictions of protein leverage. We account for the similarities and differences in performance of the two systems in an analysis of Australian foods.

Testing the protein-leverage hypothesis using population surveillance data.

It is hypothesized that humans exhibit ‘protein leverage’ (PL), whereby regulation of absolute protein intake results in the over-consumption of non-protein food on low percentage protein diets. Testing for PL using dietary surveillance data involves seeking evidence for a negative association between total energy intake and percentage energy from protein. However, it is unclear whether such an association might emerge without PL due to the structure of intake data (protein and non-protein intakes have different means and variances and covary). We derive a set of models that describe the association between the expected estimate of PL and the distributions of protein and non-protein intake. Models were validated via simulation. Patterns consistent with PL will not emerge simply because protein intake has a lower mean and/or variance than non-protein. Rather, evidence of PL is observed where protein has a lower index of dispersion (variance/mean) than non-protein intake. Reciprocally, the stronger PL is the lower the index of dispersion for protein intake becomes. Disentangling causality is ultimately beyond the power of observational data alone. However, we show that one can correct for confounders (e.g. age) in generating signals of PL, and describe independent measures that can anchor inferences around the role of PL.

Macronutrient Ratio Modification in a Semi‐Purified Diet Composition: Effects on Growth and Body Composition of Juvenile Zebrafish Danio rerio

AbstractThe interaction of dietary macronutrients in the control of growth, body composition, health, and longevity has received renewed attention. The protein leverage hypothesis proposes an inverse relationship between dietary protein levels and obesity development (low protein promoting high obesity), although the majority of laboratory studies have utilized a design modulating the protein amount within a single dietary protein source composition. We investigated whether varying the levels of dietary protein amount and sources would have impacts on growth and body composition using the Zebrafish Danio rerio model. At 28 d postfertilization, Zebrafish were fed 1 of 12 dietary treatments for 12 weeks. Diets contained different protein sources (fish protein hydrolysate [FPH], soy protein isolate [SOY], casein [CAS], or a mixture of all three sources including wheat gluten [MIX]). Sources were formulated at three protein concentrations (18, 33, or 48% as fed, substituted with wheat starch for caloric balance). Body length, height, and weight were measured over time and at termination. Contributions of macronutrients to growth and body fat outcomes were estimated by LASSO (least absolute shrinkage and selection operator) regression. Male and female length, height, and weight increased significantly in response to increasing dietary protein. Male and female fish fed SOY or FPH had the highest amount of body lipid at all protein concentrations relative to those fed CAS or MIX. Relative body lipid was highest in fish that were fed the lowest protein concentration. These data suggest an important role of macronutrient balances, including dietary protein amount and source, in relation to the protein: energy (carbohydrate and lipid) ratio on growth and body composition outcomes. These outcomes may ultimately reflect metabolic alterations that can lead to confounding interpretations of health and disease status. Furthermore, these data reinforce the need to consider and report dietary composition in establishing rigorous and reproducible nutritional guidelines for Zebrafish.

Very-low-protein diets lead to reduced food intake and weight loss, linked to inhibition of hypothalamic mTOR signaling, in mice

The protein leverage hypothesis predicts that low dietary protein should increase energy intake and cause adiposity. We designed 10 diets varying from 1% to 20% protein combined with either 60% or 20% fat. Contrasting the expectation, very low protein did not cause increased food intake. Although these mice had activated hunger signaling, they ate less food, resulting in decreased body weight and improved glucose tolerance but not increased frailty, even under 60% fat. Moreover, they did not show hyperphagia when returned to a 20% protein diet, which could be mimicked by treatment with rapamycin. Intracerebroventricular injection of AAV-S6K1 significantly blunted the decrease in both food intake and body weight in mice fed 1% protein, an effect not observed with inhibition of eIF2a, TRPML1, and Fgf21 signaling. Hence, the 1% protein diet induced decreased food intake and body weight via a mechanism partially dependent on hypothalamic mTOR signaling.

Food intake control and body weight regulation by dietary protein

Summary The protein requirement is generally defined as the amount necessary to maintain the body's protein pool. However, under free choice conditions, animal models often ingest more protein than required for nitrogen balance (10%–15%). This behavior possibly reflects the search for a high protein-to-carbohydrate ratio (0.6–0.8), inducing metabolic benefits. This indicates that in addition to protein homeostasis, dietary proteins are also involved in energy homeostasis. The mechanisms controlling protein and energy intake are partly independent and in specific conditions, there may be a conflict between the two. Protein density in the human diet has decreased ∼2% since the 1970s and, according to the protein leverage hypothesis, this decrease may be responsible for the increase in energy intake and prevalence of obesity observed today.

Cardio-metabolic consequences of dietary carbohydrates: reconciling contradictions using nutritional geometry.

Carbohydrates are the major source of dietary energy, but their role in health and disease remains controversial. Recent epidemiological evidence suggests that the increased consumption of carbohydrates is associated with obesity and increased risk of mortality and dietary trials show that carbohydrate restriction leads to weight loss and improved glycaemic status in obese and diabetic subjects. In contrast, the diets of populations with long and healthy lifespans (e.g. traditional Okinawans from Japan) are high in carbohydrate and low in protein, and several clinical and preclinical studies have linked low-carbohydrate-high-protein diets with increased mortality risk. In this paper we attempt to reconcile these contradictory findings by moving beyond traditional single-nutrient analyses to consider the interactions between nutrients on health outcomes. We do so using the Geometric Framework (GF), a nutritional modelling platform that explicitly considers the main and interactive effects of multiple nutrients on phenotypic characteristics. Analysis of human data by GF shows that weight loss and improved cardio-metabolic outcomes under carbohydrate restriction derive at least in part from reduced caloric intake due to the concomitantly increased proportion of protein in the diet. This is because, as in many animals, a specific appetite for protein is a major driver of food intake in humans. Conversely, dilution of protein in the diet leverages excess food intake through compensatory feeding for protein ('protein leverage'). When protein is diluted in the diet by readily digestible carbohydrates and fats, as is the case in modern ultra-processed foods, protein leverage results in excess calorie intake, leading to rising levels of obesity and metabolic disease. However, when protein is diluted in the diet by increased quantities of less readily digestible forms of carbohydrate and fibre, energy balance is maintained and health benefits accrue, especially during middle age and early late-life. We argue that other controversies in carbohydrate research can be resolved using the GF methodology in dietary studies.