Determining the metabolic effects of dietary fat, sugars and fat-sugar interaction using nutritional geometry in a dietary challenge study with male mice.

The metabolic effects of sugars and fat lie at the heart of the "carbohydrate vs fat" debate on the global obesity epidemic. Here, we use nutritional geometry to systematically investigate the interaction between dietary fat and the major monosaccharides, fructose and glucose, and their impact on body composition and metabolic health. Male mice (n = 245) are maintained on one of 18 isocaloric diets for 18-19 weeks and their metabolic status is assessed through in vivo procedures and by in vitro assays involving harvested tissue samples. We find that in the setting of low and medium dietary fat content, a 50:50 mixture of fructose and glucose (similar to high-fructose corn syrup) is more obesogenic and metabolically adverse than when either monosaccharide is consumed alone. With increasing dietary fat content, the effects of dietary sugar composition on metabolic status become less pronounced. Moreover, higher fat intake is more harmful for glucose tolerance and insulin sensitivity irrespective of the sugar mix consumed. The type of fat consumed (soy oil vs lard) does not modify these outcomes. Our work shows that both dietary fat and sugars can lead to adverse metabolic outcomes, depending on the dietary context. This study shows how the principles of the two seemingly conflicting models of obesity (the "energy balance model" and the "carbohydrate insulin model") can be valid, and it will help in progressing towards a unified model of obesity. The main limitations of this study include the use of male mice of a single strain, and not testing the metabolic effects of fructose intake via sugary drinks, which are strongly linked to human obesity.

Impact of dietary carbohydrate type and protein-carbohydrate interaction on metabolic health.

Reduced protein intake, through dilution with carbohydrate, extends lifespan and improves mid-life metabolic health in animal models. However, with transition to industrialised food systems, reduced dietary protein is associated with poor health outcomes in humans. Here we systematically interrogate the impact of carbohydrate quality in diets with varying carbohydrate and protein content. Studying 700 male mice on 33 isocaloric diets, we find that the type of carbohydrate and its digestibility profoundly shape the behavioural and physiological responses to protein dilution, modulate nutrient processing in the liver and alter the gut microbiota. Low (10%)-protein, high (70%)-carbohydrate diets promote the healthiest metabolic outcomes when carbohydrate comprises resistant starch (RS), yet the worst outcomes were with a 50:50 mixture of monosaccharides fructose and glucose. Our findings could explain the disparity between healthy, high-carbohydrate diets and the obesogenic impact of protein dilution by glucose-fructose mixtures associated with highly processed diets.

Rapid behavioural gregarization in the desert locust, Schistocerca gregaria entails synchronous changes in both activity and attraction to conspecifics.

Desert Locusts can change reversibly between solitarious and gregarious phases, which differ considerably in behaviour, morphology and physiology. The two phases show many behavioural differences including both overall levels of activity and the degree to which they are attracted or repulsed by conspecifics. Solitarious locusts perform infrequent bouts of locomotion characterised by a slow walking pace, groom infrequently and actively avoid other locusts. Gregarious locusts are highly active with a rapid walking pace, groom frequently and are attracted to conspecifics forming cohesive migratory bands as nymphs and/or flying swarms as adults. The sole factor driving the onset of gregarization is the presence of conspecifics. In several previous studies concerned with the mechanism underlying this transformation we have used an aggregate measure of behavioural phase state, Pgreg, derived from logistic regression analysis, which combines and weights several behavioural variables to characterise solitarious and gregarious behaviour. Using this approach we have analysed the time course of behavioural change, the stimuli that induce gregarization and the key role of serotonin in mediating the transformation. Following a recent critique that suggested that using Pgreg may confound changes in general activity with genuine gregarization we have performed a meta-analysis examining the time course of change in the individual behaviours that we use to generate Pgreg. We show that the forced crowding of solitarious locusts, tactile stimulation of the hind femora, and the short-term application of serotonin each induce concerted changes in not only locomotion-related variables but also grooming frequency and attraction to other locusts towards those characteristic of long-term gregarious locusts. This extensive meta-analysis supports and extends our previous conclusions that solitarious locusts undergo a rapid behavioural gregarization upon receiving appropriate stimulation for a few hours that is mediated by serotonin, at the end of which their behaviour is largely indistinguishable from locusts that have been in the gregarious phase their entire lives.

Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust.

BACKGROUND: The Australian plague locust, Chortoicetes terminifera, is among the most promising species to unravel the suites of genes underling the density-dependent shift from shy and cryptic solitarious behaviour to the highly active and aggregating gregarious behaviour that is characteristic of locusts. This is because it lacks many of the major phenotypic changes in colour and morphology that accompany phase change in other locust species. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method available for determining changes in gene expression. However, to accurately monitor the expression of target genes, it is essential to select an appropriate normalization strategy to control for non-specific variation between samples. Here we identify eight potential reference genes and examine their expression stability at different rearing density treatments in neural tissue of the Australian plague locust. RESULTS: Taking advantage of the new orthologous DNA sequences available in locusts, we developed primers for genes encoding 18SrRNA, ribosomal protein L32 (RpL32), armadillo (Arm), actin 5C (Actin), succinate dehydrogenase (SDHa), glyceraldehyde-3P-dehydrogenase (GAPDH), elongation factor 1 alpha (EF1a) and annexin IX (AnnIX). The relative transcription levels of these eight genes were then analyzed in three treatment groups differing in rearing density (isolated, short- and long-term crowded), each made up of five pools of four neural tissue samples from 5th instar nymphs. SDHa and GAPDH, which are both involved in metabolic pathways, were identified as the least stable in expression levels, challenging their usefulness in normalization. Based on calculations performed with the geNorm and NormFinder programs, the best combination of two genes for normalization of gene expression data following crowding in the Australian plague locust was EF1a and Arm. We applied their use to studying a target gene that encodes a Ca2+ binding glycoprotein, SPARC, which was previously found to be up-regulated in brains of gregarious desert locusts, Schistocerca gregaria. Interestingly, expression of this gene did not vary with rearing density in the same way in brains of the two locust species. Unlike S. gregaria, there was no effect of any crowding treatment in the Australian plague locust. CONCLUSION: Arm and EF1a is the most stably expressed combination of two reference genes of the eight examined for reliable normalization of RT-qPCR assays studying density-dependent behavioural change in the Australian plague locust. Such normalization allowed us to show that C. terminifera crowding did not change the neuronal expression of the SPARC gene, a gregarious phase-specific gene identified in brains of the desert locust, S. gregaria. Such comparative results on density-dependent gene regulation provide insights into the evolution of gregarious behaviour and mass migration of locusts. The eight identified genes we evaluated are also candidates as normalization genes for use in experiments involving other Oedipodinae species, but the rank order of gene stability must necessarily be determined on a case-by-case basis.

Behavioural phase change in the Australian plague locust, Chortoicetes terminifera, is triggered by tactile stimulation of the antennae.

Density-dependent phase polyphenism is a defining characteristic of the paraphyletic group of acridid grasshoppers known as locusts. The cues and mechanisms associated with crowding that induce behavioural gregarization are best understood in the desert locust, Schistocerca gregaria, and involve a combination of sensory inputs from the head (visual and olfactory) and mechanostimulation of the hind legs, acting via a transient increase in serotonin in the thoracic ganglia. Since behavioural gregarization has apparently arisen independently multiple times within the Acrididae, the important question arises as to whether the same mechanisms have been recruited each time. Here we explored the roles of visual, olfactory and tactile stimulation in the induction of behavioural gregarization in the Australian plague locust, Chortoicetes terminifera. We show that the primary gregarizing input is tactile stimulation of the antennae, with no evidence for an effect of visual and olfactory stimulation or tactile stimulation of the hind legs. Our results show that convergent behavioural responses to crowding have evolved employing different sites of sensory input in the Australian plague locust and the desert locust.

Swarm formation in the desert locust Schistocerca gregaria: isolation and NMR analysis of the primary maternal gregarizing agent.

Depending on their rearing density, female desert locusts Schistocerca gregaria epigenetically endow their offspring with differing phenotypes. To identify the chemical basis for such maternal transmission, we compared solitarious and gregarious locust egg pod foam using high performance liquid chromatography (HPLC). We revealed causal relationships between foam chemistry and hatchling phenotype (phase state) by iteratively applying HPLC fractions from gregarious locust egg foam extracts to solitarious eggs and assessing resulting hatchlings with a behavioural bioassay involving logistic regression. Selection and application of increasingly specific HPLC fractions allowed us to isolate compounds with gregarizing properties. Hatchling gregarization was triggered only by certain fractions and was dose dependent. In a final series of experiments, we characterized the most specific gregarizing fraction by nuclear magnetic resonance (NMR) spectroscopy. Here we present tentative structural features of the primary locust maternal gregarizing agent, which appears to be an alkylated l-dopa analogue. In addition, we propose a mechanism for phase-dependent regulation of this compound's activity.