Kari Ruohonen

The Ratio of Macronutrients, Not Caloric Intake, Dictates Cardiometabolic Health, Aging, and Longevity in Ad Libitum-Fed Mice

The fundamental questions of what represents a macronutritionally balanced diet and how this maintains health and longevity remain unanswered. Here, the Geometric Framework, a state-space nutritional modeling method, was used to measure interactive effects of dietary energy, protein, fat, and carbohydrate on food intake, cardiometabolic phenotype, and longevity in mice fed one of 25 diets ad libitum. Food intake was regulated primarily by protein and carbohydrate content. Longevity and health were optimized when protein was replaced with carbohydrate to limit compensatory feeding for protein and suppress protein intake. These consequences are associated with hepatic mammalian target of rapamycin (mTOR) activation and mitochondrial function and, in turn, related to circulating branched-chain amino acids and glucose. Calorie restriction achieved by high-protein diets or dietary dilution had no beneficial effects on lifespan. The results suggest that longevity can be extended in ad libitum-fed animals by manipulating the ratio of macronutrients to inhibit mTOR activation.

Dephytinization of two soy proteins increases phosphorus and protein utilization by rainbow trout, Oncorhynchus mykiss

Abstract The efficacy of pre-treating two soy proteins with phytase (dephytinization) was evaluated in 2×3 factorial trial with rainbow trout [two soy proteins, three treatments (no treatment, dephytinization, P supplement)]. Two commercial soy proteins, Soycomil P and Hamlet Protein 300, were fed with no supplemental phosphorus (P), or with 3 g supplemental P kg−1 diet. Experimental dephytinized batches of the soy proteins were prepared by wet incubation with microbial phytase (BASF Natuphos) and included in diets without supplemental P. The soy product contents were 600 g kg−1 diets. P contents were 6–9 g kg−1 in diets with and without supplemental P, respectively. Initially, 57-g fish were fed in triplicate tanks for 9 weeks. Dephytinization had a positive effect on weight gain, feed efficiency and on protein, P, calcium, magnesium and zinc utilization. Weight gain was better in fish fed dephytinized soy proteins than in fish fed untreated regular soy fortified with P. P retention averaged 33% in fish fed regular soy proteins and 72% in fish fed dephytinized soy protein. P load into the water from fish fed diets with 60% dephytinized soy proteins averaged 1.5 g P kg−1 weight gain, compared to 4.3 g P kg−1 weight gain in diets with untreated soy proteins. Although similarly low P loads cannot yet be expected from practical diets, dephytinization of soy with phytase could offer a powerful tool for nutrient discharge management in aquaculture.

Macronutrient balance, reproductive function, and lifespan in aging mice

Significance A fundamental tenet of life-history theory is that reproduction and longevity trade off against one another. Experiments on invertebrates show that, rather than competing for limiting resources, reproduction and lifespan are optimized on different dietary macronutrient compositions. In mice, studies have yet to establish the relationship between macronutrient balance, reproduction, and lifespan. We evaluated the effects of macronutrients and energy on lifespan and reproductive function. Indicators of reproductive function (uterine mass, ovarian follicle number, testes mass, epididymal sperm counts) were optimized by high protein (P), low carbohydrate (C) diets whereas lifespan was greatest on low P:C diets. Corpora lutea and estrous cycling were higher in females on lower P:C diets. Macronutrient balance has profound and opposing effects on reproduction and longevity. 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).

Effects of feeding frequency on growth and food utilisation of rainbow trout (Oncorhynchus mykiss) fed low-fat herring or dry pellets

Abstract One-year-old rainbow trout (weight range 400–700 g) were fed for 18 weeks on low-fat herring or commercial dry pellets 1, 2 or 4 times in a day. Quadratic regression analysis indicated that at least three feedings were required for maximum growth and that fish fed a dry diet could benefit from even more frequent feeding. The proportion of lipid in growth increased with increased number of feedings, but the protein content was not affected (mean 18.6% protein wet basis). Food consumption (dry weight) was affected in a similar manner for both diets and its maximum occurred at slightly higher frequency than for maximum growth. No effects of feeding frequency or type of diet on energy retention efficiency were observed (mean 42.5% of gross energy) but protein efficiency decreased with more frequent feedings. Dry diet protein was retained more efficiently than protein from herring. It is suggested that the number of feedings required for maximum growth may be dependent on the nutrient density of diet.

Optimal diet composition for European whitefish (Coregonus lavaretus): carbohydrate stress and immune parameter responses

Abstract A feeding trial was conducted on the European whitefish to study the effects of replacing fish meal with fish oil and corn starch on the stress response and immune system parameters. Nine diets with varying levels of fish meal (FM; 38–86%), fish oil (FO; 2–22%) and corn starch (CS; 0–33%), and fixed levels of wheat meal (10%) and vitamin–mineral premix (2%) were formulated and replicates were allocated among 25 tanks following the D-optimality criteria. Fish were fed the extruded diets to satiation for 10 weeks in a flow-through freshwater system at 15 °C. The liver and plasma were sampled at the termination of the trial, and the response surfaces were modeled as Scheffe polynomials specific for mixture designs. Liver glycogen and plasma glucose increased and plasma IgM decreased with increasing CS level. Plasma lysozyme levels decreased with an increase in FM. Plasma cortisol showed a linear response, with the highest values in high-FO, low-CS diets. Mortalities only occurred with high-CS diets. After the feeding trial, the effects of rapid water cooling on stress responses were measured in fish fed either a low-starch diet (66% FM, 22% FO, and 0% CS) or a high-starch diet (53% FM, 2% FO, and 33% CS). Initial plasma samples were collected at 15 °C, whereafter the temperature was reduced to 2 °C in 24 h. Plasma samples were withdrawn, and sampling was repeated after three more days at 2 °C. Cortisol increased from an initial 7 to a peak value of 70 ng ml −1 at the second sampling, and decreased thereafter to 20–30 ng ml −1 . Plasma glucose increased from an initial 3.9 to 4.8 mmol l −1 after 24 h, and further increased to approximately 7.8 mmol l −1 after three more days in cold water. The responses did not differ significantly between fish fed low-starch or high-starch diets. On overall, whitefish showed inadequate adaptation to high dietary carbohydrate levels.

Growth, bone mineralization, and heat and low oxygen tolerance in European whitefish (Coregonus lavaretus L.) fed with graded levels of phosphorus

Abstract Trials were conducted to determine the dietary phosphorus (P) requirement of European whitefish, and to assess the effects of sub-optimal P feeding on growth, bone mineralization, lipid accumulation in the body and on heat and low-oxygen tolerance. Four replicate groups of whitefish fingerlings were fed in excess with one of five semipurified diets containing 4.4 to 14.9 g P kg−1. The P requirement was estimated by nonlinear curve fitting to bone mineralization and growth rate data. Dietary P and energy availabilities were measured at the determined requirement level. Fish fed no P supplement showed typical hypophosphatemia in poorly mineralized bones, a low whole-body P content and a low plasma phosphate concentration. Hypophosphatemic fish grew slower and had elevated whole body lipid concentrations. To obtain 95% of the maximum vertebra ash concentration and maximum growth, respectively, 6.5- and 6.2-g available P kg−1 diet was required. Fish fed low dietary P contents had higher whole body lipid levels than fish fed P at or above the determined requirement level. Hypophosphatemic fish had slightly, although statistically significant, lower upper lethal temperature than fish fed with adequate P contents (28.8 vs. 29.4 °C). Tolerance of low dissolved oxygen did not differ between fish fed low or high P contents (1.47 vs. 1.45 mg l−1).

Defining the Nutritional and Metabolic Context of FGF21 Using the Geometric Framework

Fibroblast growth factor 21 (FGF21) is the first known endocrine signal activated by protein restriction. Although FGF21 is robustly elevated in low-protein environments, increased FGF21 is also seen in various other contexts such as fasting, overfeeding, ketogenic diets, and high-carbohydrate diets, leaving its nutritional context and physiological role unresolved and controversial. Here, we use the Geometric Framework, a nutritional modeling platform, to help reconcile these apparently conflicting findings in mice confined to one of 25 diets that varied in protein, carbohydrate, and fat content. We show that FGF21 was elevated under low protein intakes and maximally when low protein was coupled with high carbohydrate intakes. Our results explain how elevation of FGF21 occurs both under starvation and hyperphagia, and show that the metabolic outcomes associated with elevated FGF21 depend on the nutritional context, differing according to whether the animal is in a state of under- or overfeeding.

Optimal diet composition for European whitefish (Coregonus lavaretus): analysis of growth and nutrient utilisation in mixture model trials

Abstract Two feed mixture trials were conducted on the European whitefish. For both trials, nine diets with varying levels of fish meal (FM), fish oil (FO) and cornstarch (CS), and fixed levels of wheat meal and vitamin–mineral premix (12%), were formulated according to d -optimality criteria assuming a quadratic response surface. In the two trials, the proportions of the ingredients were, respectively: 38–86% and 43–85% for FM, 2–22% and 0–30% for FO, and 0–33% and 0–15% for CS. The extruded diets were fed to satiation for 72 and 85 days in a flow-through, freshwater system at 15 °C. Optimal allocation of replicates among 25 and 29 tanks was based on d -optimality criteria. The response surfaces were modelled as quadratic and super-cubic polynomials specific for mixture designs. The data from the trials were combined for the final analysis, which included the experiment as a random effect. Fish growth increased with increasing FM content up to 66.3% FM (16.7% FO and 5.0% CS). Protein growth peaked at a somewhat higher FM content of 72.0% (10.2% FO, 5.9% CS), and lipid deposition rate at the highest studied FO content (53.1% FM, 30.0% FO and 4.9% CS). Feed efficiency improved with decreasing CS content and the highest value was obtained with the mixture containing 58.0% FM, 30.0% FO and 0.0% CS. Protein retention efficiency was improved with decreasing FM content and peaked at a mixture of 50.2% FM, 30.0% FO and 7.8% CS. The diet inducing 95% of the maximum growth with the minimum FM content comprised of 51.2% FM, 30.0% FO and 6.8% CS. Based on the data from these two experiments, an area of optimal diet formulation can be assessed by graphically superimposing isobars of 95% growth, feed efficiency and dietary protein utilisation responses. This optimal composition for the European whitefish appears to be in the range of 53–61% FM, 24–30% FO and 0–7% CS together with the 12% wheat meal and vitamin–mineral premix. However, this optimisation is based on round weight and because high dietary lipid levels induced high lipid deposition rates, the practical optimum for dressed weight would be at a somewhat lower FO level. Mixture experimentation proved to be an efficient method to rapidly screen European whitefish diets for nutritional optimisation.

Individual measurements and nested designs in aquaculture experiments: a simulation study

Simple and nested models for analysis of variance (ANOVA) in aquaculture experiments were compared with the help of computer simulations. Simple models for analysing variables that are based on tank means, such as final weight and growth rate, were found to be sensitive to differences in the number of individual observations in each tank. In comparison to nested models that take into account individual measurements, the simple models were found to overestimate the F ratio and increase the risk of committing type I error, i.e., accepting a false alternative hypothesis. Further, nested models permit greater flexibility in experimental design, and allow more economical solutions within a given set of constraints and required statistical power. It is concluded that individual measurements do not remove the necessity of having replicate tanks but maintaining both levels of variation (tank and individual) in the statistical analysis could reduce the number of replicates needed.

The Influence of Macronutrients on Splanchnic and Hepatic Lymphocytes in Aging Mice

There is a strong association between aging, diet, and immunity. The effects of macronutrients and energy intake on splanchnic and hepatic lymphocytes were studied in 15 month old mice. The mice were ad-libitum fed 1 of 25 diets varying in the ratios and amounts of protein, carbohydrate, and fat over their lifetime. Lymphocytes in liver, spleen, Peyers patches, mesenteric lymph nodes, and inguinal lymph nodes were evaluated using flow cytometry. Low protein intake reversed aging changes in splenic CD4 and CD8 T cells, CD4:CD8 T cell ratio, memory/effector CD4 T cells and naïve CD4 T cells. A similar influence of total caloric intake in these ad-libitum fed mice was not apparent. Protein intake also influenced hepatic NK cells and B cells, while protein to carbohydrate ratio influenced hepatic NKT cells. Hepatosteatosis was associated with increased energy and fat intake and changes in hepatic Tregs, effector/memory T, and NK cells. Hepatic NK cells were also associated with body fat, glucose tolerance, and leptin levels while hepatic Tregs were associated with hydrogen peroxide production by hepatic mitochondria. Dietary macronutrients, particularly protein, influence splanchnic lymphocytes in old age, with downstream associations with mitochondrial function, liver pathology, and obesity-related phenotype.