Metabolic effects of the dietary monosaccharides fructose, fructose-glucose, or glucose in mice fed a starch-containing moderate high-fat diet

Fructose consumption has been linked to obesity and increased hepatic de novo lipogenesis (DNL). Excessive caloric intake often confounds the results of fructose studies, and experimental diets are generally low-fat diets, not representative for westernized diets. Here, we compared the effects of dietary fructose with those of dietary glucose, in adult male and female mice on a starch-containing moderate high-fat (HF) diet. After 5 weeks fattening on a HF high-glucose (HF-G) diet, mice were stratified per sex and assigned to one of the three intervention diets for 6 weeks: HF high fructose (HF-F), HF with equimolar glucose and fructose (HF-GF), or HF-G. Bodyweight (BW) and food intake were measured weekly. Indirect calorimetry was performed on week 5; animals were sacrificed in food-deprived state on week 6. Data were analyzed within sex. BW gain was similar among animals on the HF-G, HF-GF, and HF-F diets. Cumulative food intake was slightly lower in HF-F animals (both sexes). However, energy expenditure was not affected, or were circulating insulin and glucose concentrations, and hepatic triglyceride levels at endpoint. Hepatic gene expression analysis showed only minor alterations in hexokinase and glycolysis-related expression in males, and no alterations in sugar transporters, or DNL-related enzymes. In females, no consistent alterations in hepatic or small intestine gene expression were seen. Concluding, partial or complete replacement of dietary glucose with fructose does not increase caloric intake, and does not affect BW, hepatic triglyceride levels, or insulin concentrations in male and female mice on a moderate high-fat diet.</p

Post-weaning metabolic programming by dietary monosaccharides

Nutrition in early life can have lasting effects on metabolism: nutritional programming. Pregnancy and lactation are well established critical periods of development impacting lifelong health, but development continues after these periods. Therefore, nutrition in later periods, such as the weaning and (early) post-weaning period, may also induce lasting metabolic effects. After lactation, when an infant relies solely on milk for nutrition, the weaning period commences. In this phase, solid foods are gradually introduced; it is a period of great diversification of the diet. The transition to solid foods introduces a major change in carbohydrate exposure. The main carbohydrate in the lactation period is lactose, a dimer of glucose and galactose. Gradually, a variety of di- and polysaccharides are introduced that are mainly glucose based, but can also contain fructose. Whether or not exposure to different types of monosaccharides at weaning has lasting effects on metabolism and metabolic health is not known. The aim of this thesis was to establish whether the post-weaning period is a critical developmental period in which exposure to different types of monosaccharides can lead to programming of adult metabolic health. We were particularly interested in galactose and fructose. Our interest in galactose was because this sugar almost disappears from the diet after weaning. Our interest in fructose was because it appears in the diet at weaning, and is suggested to be more detrimental to health compared to glucose, which is always present and is taken as a reference. Thus we compared effects of the dietary monosaccharides fructose and galactose, to those of glucose. Analyses were focussed on body composition and metabolic health. A mouse model for nutritional programming was used. In this model, newly weaned C57BL/6JRccHsd mice were fed with diets differing in monosaccharide content for three weeks. Afterwards, all mice were fed an obesogenic high-fat diet (HFD) for nine weeks. Chapter 2 describes a mouse study where the effects of fructose in the post-weaning diet on later life health were compared to the effects of glucose alone. Body weight, body composition, and organ weights were similar in both groups after the nine-week HFD period. Indirect calorimetry analyses indicated that respiratory exchange ratio&rsquo;s (RER&rsquo;s), energy expenditure, activity, and metabolic flexibility were not different between fructose- and glucose fed animals, neither directly, nor when on the HFD. Serum insulin concentrations were significantly lower in females fed fructose post weaning, than in females fed glucose post weaning, while serum insulin concentrations were not significantly affected in males. From these data we concluded that fructose and glucose are comparable in their direct effect, and there is no adverse programming of fructose compared to glucose in the post-weaning period. For additional insight in metabolic effects of fructose, the direct effects of fructose, fructose and glucose in a 1:1 ratio, and glucose, were studied in adult male and female mice on a moderate HFD (Chapter 3). Mice on the HFD with fructose had slightly lower energy intakes overall. Body weight was not affected by the monosaccharide composition of the diet, and nor were plasma insulin concentrations. Hepatic gene expression analyses showed minor upregulation of hexokinase expression in fructose fed compared to glucose fed males, without significant alterations in sugar transporters, or glycolysis- or de novo lipogenesis-related enzymes. Gene expression in the liver and intestine of female mice showed no consistent differences. Overall, our physiological data indicate that isocaloric dietary fructose does not result in more adverse physiological effects than a diet containing glucose-fructose or glucose. Next, we examined the effects of galactose. The programming effect of post-weaning galactose and glucose in a 1:1 ratio mimicking the milk sugar lactose was compared to the effect of glucose alone (Chapter 4). In females, body weight and fat mass gain on the HFD were significantly lower in animals fed galactose post weaning. These females had lower circulating serum insulin concentrations, lower adipose depots weights, with a tendency towards smaller adipocytes in gonadal white adipose tissue, and altered insulin-signalling-related gene expression. Although food intake was significantly higher in the post-weaning period, and significantly lower in the HFD period, no effects in hypothalamic gene expression on food-intake related neuropeptides or leptin signalling were found. In males, fat mass development was not affected by post-weaning monosaccharides. Concluding, this study showed that replacing glucose with galactose in a post-weaning diet, in a 1:1 ratio (mimicking lactose), had beneficial metabolic programming effects in female mice, over glucose alone. Chapter 5 shows the direct effect of post-weaning galactose and glucose in a 1:1 ratio compared to glucose alone in females. Females on the galactose diet had a higher food intake and a two-fold higher drink intake than females on the glucose diet. High-performance anion-exchange chromatography analyses indicated galactose presence in the urine of females on the galactose diet. Indirect calorimetry measurements showed no significant effect on energy expenditure or average RER, but maximal RER in the dark phase was lower in females on the galactose diet. Serum insulin concentrations and hepatic triglyceride levels were lower in females on the galactose diet. Transcriptomic analysis of the liver indicated that the gene expression profiles in metabolic pathways were not significantly affected by the diet, but inflammation-related gene expression profiles were slightly downregulated in galactose-fed females. Concluding, replacing part of glucose with galactose in the post-weaning diet reduces hepatic TG content and hepatic inflammation, implying immediate beneficial effects. In a second mouse study examining the metabolic programming effects of post-weaning galactose in females, presented in Chapter 6, no differences in body weight gain and fat mass gain were seen in the HFD period. The oral glucose tolerance test showed no difference in glucose tolerance, but indicated that circulating plasma insulin concentrations were relatively more increased in females fed galactose as analysed by the insulin incremental area under the curve. At the end of the study, no significant differences were found in circulating insulin concentrations, AKT phosphorylation and insulin-related gene expression in gonadal white adipose tissue. Concluding, replacing part of glucose with galactose in the post-weaning diet did not beneficially affect body composition or insulin signalling in adult female mice in an obesogenic environment in this study. Differences between the results of Chapter 4 and Chapter 6 may be due to differences in the experimental conditions. In Chapter 7 the findings of this thesis are discussed. Concluding, this thesis shows that the post-weaning period may be susceptible for nutritional programming by dietary monosaccharides, in particular by galactose. The effect is modest, and inconclusive as two studies yielded different outcomes. Although there are some clues that insulin signalling is involved, it is so far not clear what the main mechanism is. Females seem to be more susceptible to programming by monosaccharides in the post-weaning period than males. Our results suggest that there are no major differences in metabolic programming by fructose and glucose in the post-weaning period. Also no major differences were seen between fructose and glucose in adult mice. Extending the period of galactose intake after weaning may be beneficial as it seems to protect against liver inflammation

Partial replacement of glucose with galactose in the post-weaning diet positively affects markers of liver health

Previous research showed a beneficial programming effect of replacting glucose in the post-weaning diet with galactose on later life adiposity. Here, we studied the direct effect of the diets in the postweaning phase in female mice. In this study, female mice were fed a glucose diet (32 en% glucose; GLU) or a glucose+galactose diet (16 en% glucose and 16 en% galactose; GLU+GAL) postweaning for three weeks, from postnatal day (PN) 21 till PN42. We observed lower circulating insulin levels and lower hepatic triglyceride levels in the females on the GLU+GAL diet. The body weight, fat mass, liver weight and liver glycogen content did not differ between the groups. We next studied hepatic gene expression profiles, because of the altered hepatic triglyceride levels and since the liver is considered the primary site of galactose metabolism. However, detailed analyses including pathway analysis, showed mainly inflammation being reduced by the GLU+GAL treatment. This was confirmed by qPCR of liver tissues and focussed serum protein analysis

Partial replacement of glucose with galactose in the post-weaning diet positively affects markers of liver health

Previous research showed a beneficial programming effect of replacting glucose in the post-weaning diet with galactose on later life adiposity. Here, we studied the direct effect of the diets in the postweaning phase in female mice. In this study, female mice were fed a glucose diet (32 en% glucose; GLU) or a glucose+galactose diet (16 en% glucose and 16 en% galactose; GLU+GAL) postweaning for three weeks, from postnatal day (PN) 21 till PN42. We observed lower circulating insulin levels and lower hepatic triglyceride levels in the females on the GLU+GAL diet. The body weight, fat mass, liver weight and liver glycogen content did not differ between the groups. We next studied hepatic gene expression profiles, because of the altered hepatic triglyceride levels and since the liver is considered the primary site of galactose metabolism. However, detailed analyses including pathway analysis, showed mainly inflammation being reduced by the GLU+GAL treatment. This was confirmed by qPCR of liver tissues and focussed serum protein analysis

An exploration of the potential contribution of genetic modification and genome editing to the development of abiotic stress-tolerant crops as compared to conventional breeding

COGEM has commissioned a research project to obtain insight in the possibilities that gene-editing may offer for the development of abiotic stress tolerant crops. Genetically modified abiotic stress tolerant crops were also included in the research project. The project was carried out by Wageningen University & Research and Wageningen Food Safety Research.The results indicate that most of the effort is put into obtaining drought tolerant crops, followed by crops tolerant to salinity or heat. Worldwide there are a few genetically modified abiotic stress tolerant crops that have been authorised for cultivation. Several genetically modified abiotic stress tolerant crops have been tested in the field.There are only a few examples of gene edited abiotic stress tolerant crops. Results from extensive field testing with these gene edited crops have not been published yet.According to the researchers abiotic stress tolerant crops could be developed faster and more efficient using gene editing. With gene editing gene expression may be changed in a more subtle way, which might allow the development of crops that only express the genes which confer abiotic stress tolerance under stressful conditions. In addition, gene editing may be used to change several genes simultaneously, which may allow signaling routes that play a role in abiotic stress tolerance to be altered in a more balanced way thereby reducing the occurrence of unwanted negative side effects

Metabolic effects of the dietary monosaccharides fructose, fructose-glucose, or glucose in mice fed a starch-containing moderate high-fat diet

Fructose consumption has been linked to obesity and increased hepatic de novo lipogenesis (DNL). Excessive caloric intake often confounds the results of fructose studies, and experimental diets are generally low-fat diets, not representative for westernized diets. Here, we compared the effects of dietary fructose with those of dietary glucose, in adult male and female mice on a starch-containing moderate high-fat (HF) diet. After 5 weeks fattening on a HF high-glucose (HF-G) diet, mice were stratified per sex and assigned to one of the three intervention diets for 6 weeks: HF high fructose (HF-F), HF with equimolar glucose and fructose (HF-GF), or HF-G. Bodyweight (BW) and food intake were measured weekly. Indirect calorimetry was performed on week 5; animals were sacrificed in food-deprived state on week 6. Data were analyzed within sex. BW gain was similar among animals on the HF-G, HF-GF, and HF-F diets. Cumulative food intake was slightly lower in HF-F animals (both sexes). However, energy expenditure was not affected, or were circulating insulin and glucose concentrations, and hepatic triglyceride levels at endpoint. Hepatic gene expression analysis showed only minor alterations in hexokinase and glycolysis-related expression in males, and no alterations in sugar transporters, or DNL-related enzymes. In females, no consistent alterations in hepatic or small intestine gene expression were seen. Concluding, partial or complete replacement of dietary glucose with fructose does not increase caloric intake, and does not affect BW, hepatic triglyceride levels, or insulin concentrations in male and female mice on a moderate high-fat diet

Extended indirect calorimetry with isotopic CO₂ sensors for prolonged and continuous quantification of exogenous vs. total substrate oxidation in mice

Indirect calorimetry (InCa) estimates whole-body energy expenditure and total substrate oxidation based on O2 consumption and CO2 production, but does not allow for the quantification of oxidation of exogenous substrates with time. To achieve this, we incorporated 13CO2 and 12CO2 gas sensors into a commercial InCa system and aimed to demonstrate their performance and added value. As a performance indicator, we showed the discriminative oscillations in 13CO2 enrichment associated with food intake in mice fed diets containing naturally low (wheat) vs high (maize) 13C enrichment. To demonstrate the physiological value, we quantified exogenous vs total carbohydrate and fat oxidation continuously, in real time in mice varying in fat mass. Diet-induced obese mice were fed a single liquid mixed meal containing 13C-isotopic tracers of glucose or palmitate. Over 13 h, ~70% glucose and ~48% palmitate ingested were oxidised. Exogenous palmitate oxidation depended on body fat mass, which was not the case for exogenous glucose oxidation. We conclude that extending an InCa system with 13CO2 and 12CO2 sensors provides an accessible and powerful technique for real-time continuous quantification of exogenous and whole-body substrate oxidation in mouse models of human metabolic physiology.</p

Replacing Part of Glucose with Galactose in the Postweaning Diet Protects Female But Not Male Mice from High-Fat Diet-Induced Adiposity in Later Life

BACKGROUND: Duration of breastfeeding is positively associated with decreased adiposity and increased metabolic health in later life, which might be related to galactose. OBJECTIVE: The aim of this study was to investigate if partial replacement of glucose with galactose in the postweaning diet had a metabolic programming effect. METHODS: Male and female mice (C57BL/6JRccHsd) received an isocaloric diet (16 energy% fat; 64 energy% carbohydrates; 20 energy% protein) with either glucose (32 energy%) (GLU) or glucose + galactose (GLU + GAL, 16 energy% each) for 3 wk postweaning. Afterwards, all mice were switched to the same 40 energy% high-fat diet (HFD) for 9 wk to evaluate potential programming effects in an obesogenic environment. Data were analyzed within sex. RESULTS: Female body weight (-14%) and fat mass (-47%) were significantly lower at the end of the HFD period (both P < 0.001) among those fed GLU + GAL than among those fed GLU; effects in males were in line with these findings but nonsignificant. Food intake was affected in GLU + GAL-fed females (+8% on postweaning diet, -9% on HFD) compared with GLU-fed females, but not for hypothalamic transcript levels at endpoint. Also, in GLU + GAL-fed females, serum insulin concentrations (-48%, P < 0.05) and the associated homeostasis model assessment of insulin resistance (HOMA-IR) were significantly lower ( P < 0.05) at endpoint, but there were no changes in pancreas morphology. In GLU + GAL-fed females, expression of insulin receptor substrate 2 (Irs2) (-27%, P < 0.01 ; -44%, P < 0.001) and the adipocyte size markers leptin (Lep) (-40%, P < 0.05; -63% , P < 0.05) and mesoderm-specific transcript homolog protein (Mest) (-80%, P < 0.05; -72%, P < 0.05) was lower in gonadal and subcutaneous white adipose tissue (WAT), respectively. Expression of insulin receptor substrate1 (Irs1) (-24%, P < 0.05) was only lower in subcutaneous WAT in GLU + GAL-fed females. CONCLUSIONS: Partial replacement of glucose with galactose, resulting in a 1:1 ratio mimicking lactose, in a 3-wk postweaning diet lowered body weight, adiposity, HOMA-IR, and expression of WAT insulin signaling in HFD-challenged female mice in later life. This suggests that prolonged galactose intake may improve metabolic and overall health in later life.</p

Direct and Long-Term Metabolic Consequences of Lowly vs. Highly-Digestible Starch in the Early Post-Weaning Diet of Mice

Starches of low and high digestibility have different metabolic effects. Here, we examined whether this gives differential metabolic programming when fed in the immediate post-weaning period. Chow-fed mice were time-mated, and their nests were standardized and cross-fostered at postnatal days 1⁻2. After postnatal week (PW) 3, individually housed female and male offspring were switched to a lowly-digestible (LDD) or highly-digestible starch diet (HDD) for three weeks. All of the mice received the same high-fat diet (HFD) for nine weeks thereafter. Energy and substrate metabolism and carbohydrate fermentation were studied at the end of the HDD/LDD and HFD periods by extended indirect calorimetry. Glucose tolerance (PW 11) and metabolic flexibility (PW14) were analyzed. Directly in response to the LDD versus the HDD, females showed smaller adipocytes with less crown-like structures in gonadal white adipose tissue, while males had a lower fat mass and higher whole body fat oxidation levels. Both LDD-fed females and males showed an enlarged intestinal tract. Although most of the phenotypical differences disappeared in adulthood in both sexes, females exposed to LDD versus HDD in the early post-weaning period showed improved metabolic flexibility in adulthood. Cumulatively, these results suggest that the type of starch introduced after weaning could, at least in females, program later-life health.</p

A Lowly Digestible-Starch Diet after Weaning Enhances Exogenous Glucose Oxidation Rate in Female, but Not in Male, Mice

Starches of low digestibility are associated with improved glucose metabolism. We hypothesise that a lowly digestible-starch diet (LDD) versus a highly digestible-starch diet (HDD) improves the capacity to oxidise starch, and that this is sex-dependent. Mice were fed a LDD or a HDD for 3 weeks directly after weaning. Body weight (BW), body composition (BC), and digestible energy intake (dEI) were determined weekly. At the end of the intervention period, whole-body energy expenditure (EE), respiratory exchange ratio (RER), hydrogen production, and the oxidation of an oral 13C-labelled starch bolus were measured by extended indirect calorimetry. Pancreatic amylase activity and total 13C hepatic enrichment were determined in females immediately before and 4 h after administration of the starch bolus. For both sexes, BW, BC, and basal EE and RER were not affected by the type of starch, but dEI and hydrogen production were increased by the LDD. Only in females, total carbohydrate oxidation and starch-derived glucose oxidation in response to the starch bolus were higher in LDD versus HDD mice; this was not accompanied by differences in amylase activity or hepatic partitioning of the 13C label. These results show that starch digestibility impacts glucose metabolism differently in females versus males.</p