Role of liver AMPK and GCN2 kinases in the control of postprandial protein metabolism in response to mid-term high or low protein intake in mice.

PURPOSE: Protein synthesis and proteolysis are known to be controlled through mammalian target of rapamycin, AMP-activated kinase (AMPK) and general control non-derepressible 2 (GCN2) pathways, depending on the nutritional condition. This study aimed at investigating the contribution of liver AMPK and GCN2 on the adaptation to high variations in protein intake. METHODS: To evaluate the answer of protein pathways to high- or low-protein diet, male wild-type mice and genetically modified mice from C57BL/6 background with liver-specific AMPK- or GCN2-knockout were fed from day 25 diets differing in their protein level as energy: LP (5%), NP (14%) and HP (54%). Two hours after a 1 g test meal, protein synthesis rate was measured after a 13C valine flooding dose. The gene expression of key enzymes involved in proteolysis and GNC2 signaling pathway were quantified. RESULTS: The HP diet but not the LP diet was associated with a decrease in fractional synthesis rate by 29% in the liver compared to NP diet. The expression of mRNA encoding ubiquitin and Cathepsin D was not sensitive to the protein content. The deletion of AMPK or GCN2 in the liver did not affect nor protein synthesis rates and neither proteolysis markers in the liver or in the muscle, whatever the protein intake. In the postprandial state, protein level alters protein synthesis in the liver but not in the muscle. CONCLUSIONS: Taken together, these results suggest that liver AMPK and GCN2 are not involved in this adaptation to high- and low-protein diet observed in the postprandial period.

Plasma FGF21 concentrations and spontaneous self-selection of protein suggest that 15% protein in the diet may not be enough for male adult rats.

Protein requirement has been determined at 10%-15% energy. Under dietary self-selection, rats ingest 25%-30% energy as protein and regulate FGF21 (a hormone signaling protein deficiency) to levels lower than those measured with a 15% protein (15P) diet. Our hypothesis is that if a 15P diet was indeed sufficient to ensure protein homeostasis, it is probably a too low protein level to ensure optimal energy homeostasis. Adult male Wistar rats were used in this study. The first objective was to determine the changes in food intake, body composition, and plasma FGF21, IGF-1, and PYY concentrations in rats fed 8P, 15P, 30P, 40P, or 50P diets. The second was to determine whether the FGF21 levels measured in the rats were related to spontaneous protein intake. Rats were fed a 15P diet and then allowed to choose between a protein diet and a protein-free diet. Food intake and body weight were measured throughout the experiments. Body composition was determined at different experimental stages. Plasma samples were collected to measure FGF21, IGF-1, and PYY concentrations. A 15P diet appears to result in higher growth than that observed with the 30P, 40P, and 50P diets. However, the 15P diet probably does not provide optimal progression of body composition owing to a tendency of 15P rats to fix more fat and energy in the body. The variable and higher concentrations of FGF21 in the 15P diet suggest a deficit in protein intake, but this does not appear to be a parameter reflecting the adequacy of protein intake relative to individual protein requirements.NEW & NOTEWORTHY Under dietary self-selection, rats choose to ingest 25%-30% of energy as protein, a value higher than the protein requirement (10%-15%). According to our results, this higher spontaneous intake reflects the fact that rats fed a 15% protein diet, compared with high-protein diets, tend to bind more fat and have higher concentrations of FGF21, a hormone signaling protein deficiency. A 15% protein diet appears to be sufficient for protein homeostasis but not for optimal energy homeostasis.

Lysine or Threonine Deficiency Decreases Body Weight Gain in Growing Rats despite an Increase in Food Intake without Increasing Energy Expenditure in Response to FGF21.

The objective of this study is to evaluate the effects of a strictly essential amino acid (lysine or threonine; EAA) deficiency on energy metabolism in growing rats. Rats were fed for three weeks severely (15% and 25% of recommendation), moderately (40% and 60%), and adequate (75% and 100%) lysine or threonine-deficient diets. Food intake and body weight were measured daily and indirect calorimetry was performed the week three. At the end of the experimentation, body composition, gene expression, and biochemical analysis were performed. Lysine and threonine deficiency induced a lower body weight gain and an increase in relative food intake. Lysine or threonine deficiency induced liver FGF21 synthesis and plasma release. However, no changes in energy expenditure were observed for lysine deficiency, unlike threonine deficiency, which leads to a decrease in total and resting energy expenditure. Interestingly, threonine severe deficiency, but not lysine deficiency, increase orexigenic and decreases anorexigenic hypothalamic neuropeptides expression, which could explain the higher food intake. Our results show that the deficiency in one EAA, induces a decrease in body weight gain, despite an increased relative food intake, without any increase in energy expenditure despite an induction of FGF21.

Protein-carbohydrate interaction effects on energy balance, FGF21, IGF-1, and hypothalamic gene expression in rats.

Amino acids are involved in energy homeostasis, just as are carbohydrates and lipids. Therefore, mechanisms controlling protein intake should operate independently and in combination with systems controlling overall energy intake to coordinate appropriate metabolic and behavioral responses. The objective of this study was to quantify the respective roles of dietary protein and carbohydrate levels on energy balance, plasma fibroblast growth factor 21 (FGF21) and insulin growth factor 1 (IGF-1) concentrations, and hypothalamic neurotransmitters (POMC, NPY, AgRP, and CART). In a simplified geometric framework, 7-wk-old male Wistar rats were fed 12 diets containing 3%-30% protein for 3 wk, in which carbohydrates accounted for 30%-75% of the carbohydrate and fat part of the diet. As a result of this study, most of the studied parameters (body composition, energy expenditure, plasma FGF21 and IGF-1 concentrations, and Pomc/Agrp ratio) responded mainly to the protein content and to a lesser extent to the carbohydrate content in the diet.NEW & NOTEWORTHY As mechanisms controlling protein intake can operate independently and in combination with those controlling energy intakes, we investigated the metabolic and behavioral effects of the protein-carbohydrate interaction. With a simplified geometric framework, we showed that body composition, energy balance, plasma FGF21 and IGF-1 concentrations, and hypothalamic Pomc/Agrp ratio were primarily responsive to protein content and, to a lesser extent, to carbohydrate content of the diet.

Severe protein deficiency induces hepatic expression and systemic level of FGF21 but inhibits its hypothalamic expression in growing rats.

To study, in young growing rats, the consequences of different levels of dietary protein deficiency on food intake, body weight, body composition, and energy balance and to assess the role of FGF21 in the adaptation to a low protein diet. Thirty-six weanling rats were fed diets containing 3%, 5%, 8%, 12%, 15% and 20% protein for three weeks. Body weight, food intake, energy expenditure and metabolic parameters were followed throughout this period. The very low-protein diets (3% and 5%) induced a large decrease in body weight gain and an increase in energy intake relative to body mass. No gain in fat mass was observed because energy expenditure increased in proportion to energy intake. As expected, Fgf21 expression in the liver and plasma FGF21 increased with low-protein diets, but Fgf21 expression in the hypothalamus decreased. Under low protein diets (3% and 5%), the increase in liver Fgf21 and the decrease of Fgf21 in the hypothalamus induced an increase in energy expenditure and the decrease in the satiety signal responsible for hyperphagia. Our results highlight that when dietary protein decreases below 8%, the liver detects the low protein diet and responds by activating synthesis and secretion of FGF21 in order to activate an endocrine signal that induces metabolic adaptation. The hypothalamus, in comparison, responds to protein deficiency when dietary protein decreases below 5%.

Rats Self-Select a Constant Protein-to-Carbohydrate Ratio Rather Than a Constant Protein-to-Energy Ratio and Have Low Plasma FGF21 Concentrations.

BACKGROUND: Under dietary self-selection (DSS), rats ingest 25-30% of energy as protein. This high level appears to be explained by metabolic benefits related to reduced carbohydrate dependence and associated pathologies. However, the mechanisms underlying these choices remain largely misunderstood. OBJECTIVES: The aim was to test the hypothesis that in a DSS model, rats select a protein-to-energy (PE) ratio to maintain the protein-to-carbohydrate (PC) ratio constant and that fibroblast growth factor 21 (FGF21) is involved in this response. METHODS: Adult male Wistar rats were used in 3 experiments. The first was to determine whether the PE ratio was influenced by changes in carbohydrate content. The second was to test whether the PE ratio was defended with a modified DSS model. The third was to determine whether the selected PE ratio was of metabolic interest compared with a standard 15% protein diet. Food intake, body weight, and energy expenditure were measured. After 3 wk, plasma was sampled and rats were killed to determine body composition and gene expression. Statistical analyses were mainly done by ANOVA tests and correlation tests. RESULTS: The selected PE ratio increased from 20% to 35% when the carbohydrate content of the protein-free diet increased from 30% to 75% (R2 = 0.56; P < 10-6). Consequently, the PC ratio was constant (70%) in all groups (P = 0.18). In self-selecting rats, plasma FGF21 concentrations were 3 times lower than in rats fed the 5% protein diet (P < 10-4) and similar to those in rats fed a 30% diet. CONCLUSIONS: This study showed that self-selecting rats established PE ratios larger than those considered sufficient to achieve optimal growth in adult rats (10-15%), and the ratios were highly dependent on carbohydrates, apparently with the aim of maintaining a constant and high PC ratio. This was associated with a minimization of plasma FGF21.

What does self-selection of dietary proteins in rats tell us about protein requirements and body weight control?

Omnivores are able to correctly select adequate amounts of macronutrients from natural foods as well as purified macronutrients. In the rat model, the selected protein levels are often well above the requirements estimated from the nitrogen balance. These high intake levels were initially interpreted as reflecting poor control of protein intake, but the selected levels were later found to be precisely controlled for changes in dietary protein quality and adjusted for cold, exercise, pregnancy, lactation, age, etc. and therefore met physiological requirements. Several authors have also suggested that instead of a given level of protein intake, rodents regulate a ratio of protein to dietary carbohydrates in order to achieve metabolic benefits such as reduced insulin levels, improved blood glucose control, and, in the long term, reduced weight and fat gain. The objective of this review was to analyze the most significant results of studies carried out on rats and mice since the beginning of the 20th century, to consider what these results can bring us to interpret the current causes of the obesity pandemic and to anticipate the possible consequences of policies aimed at reducing the contribution of animal proteins in the human diet.

Activation of Adenosine Monophosphate-Activated Protein Kinase Reduces the Onset of Diet-Induced Hepatocellular Carcinoma in Mice.

The worldwide obesity and type 2 diabetes epidemics have led to an increase in nonalcoholic fatty liver disease (NAFLD). NAFLD covers a spectrum of hepatic pathologies ranging from simple steatosis to nonalcoholic steatohepatitis, characterized by fibrosis and hepatic inflammation. Nonalcoholic steatohepatitis predisposes to the onset of hepatocellular carcinoma (HCC). Here, we characterized the effect of a pharmacological activator of the intracellular energy sensor adenosine monophosphate-activated protein kinase (AMPK) on NAFLD progression in a mouse model. The compound stimulated fat oxidation by activating AMPK in both liver and skeletal muscle, as revealed by indirect calorimetry. This translated into an ameliorated hepatic steatosis and reduced fibrosis progression in mice fed a diet high in fat, cholesterol, and fructose for 20 weeks. Feeding mice this diet for 80 weeks caused the onset of HCC. The administration of the AMPK activator for 12 weeks significantly reduced tumor incidence and size. Conclusion: Pharmacological activation of AMPK reduces NAFLD progression to HCC in preclinical models.

Protein Status Modulates an Appetite for Protein To Maintain a Balanced Nutritional State-A Perspective View.

Protein sufficiency is tightly controlled through different sensing and signaling processes that modulate and adapt protein and energy metabolism and feeding behavior to reach and maintain a well-balanced protein status. High-protein diets, often discussed in the context of body weight management, usually activate anorexigenic pathways, leading to higher satiety, decreased food and energy intake, and decreased body weight and adiposity. Diets marginally low in protein (3-8% energy) or marginally deficient in some indispensable amino acid more often activate orexigenic pathways, with higher appetite and a specific appetite for protein, a response that leads to an increase in protein intake to partially compensate for the deficit in protein and amino acid. Diets severely deficient in protein (2-3% energy as protein) usually depress food intake and induce lower weight and lower fat mass and lean tissues that characterize a status of protein deficiency. The control of protein sufficiency involves various peripheral and central signals, including modulation of both metabolic pathways at the periphery as well as central pathways of the control of food and protein intake, including a reward-driven specific sensitivity to the protein content of foods.

Liver GCN2 controls hepatic FGF21 secretion and modulates whole body postprandial oxidation profile under a low-protein diet.

General control nonderepressible 2 (GCN2) is a kinase that detects amino acid deficiency and is involved in the control of protein synthesis and energy metabolism. However, the role of hepatic GCN2 in the metabolic adaptations in response to the modulation of dietary protein has been seldom studied. Wild-type (WT) and liver GCN2-deficient (KO) mice were fed either a normo-protein diet, a low-protein diet, or a high-protein diet for 3 wk. During this period, body weight, food intake, and metabolic parameters were followed. In mice fed normo- and high-protein diets, GCN2 pathway in the liver is not activated in WT mice, leading to a similar metabolic profile with the one of KO mice. On the contrary, a low-protein diet activates GCN2 in WT mice, inducing FGF21 secretion. In turn, FGF21 maintains a high level of lipid oxidation, leading to a different postprandial oxidation profile compared with KO mice. Hepatic GCN2 controls FGF21 secretion under a low-protein diet and modulates a whole body postprandial oxidation profile.

Protein status modulates the rewarding value of foods and meals to maintain an adequate protein intake.

Proteins are dietary components that contribute to nutritional needs of the body through the provision of nitrogen and amino acids. Protein status is tightly and continuously controlled to prevent or counteract protein deficiency and to maintain or restore an adequate protein status. Animals learn to detect and avoid diets deficient or devoid in protein or in at least one indispensable amino acid and when given a choice reject these diets. Diets restricted marginally in protein or in one or more amino acids more often induce hyperphagia, interpreted as an attempt to increase protein or amino acid intake and to meet the need for protein and amino acids. The increase in energy intake induced by a low protein diet is compensated for by an increased energy expenditure that restrains the gain in adiposity. The status of protein and/or amino acid insufficiency induced by protein or amino acid restricted diets is characterized by a profile of peripheral and central signals that contribute to modulate peripheral metabolic adaptations and central pathways involved in the control of feeding behaviour. These processes impact on the motivation for food and food choice, with an appetite for protein and/or for the limiting amino acid (s) associated with a reward driven sensitivity to protein and amino acid content of food and diets, which leads to restore or maintain an adequate protein status. In contrast to a protein-restricted diet, high-protein diets are usually reported to decrease food intake in both animals and humans, at least for a transient period, in relation to a reported satiating effect of proteins through activation of anorexigenic pathways.

High Pancreatic Amylase Expression Promotes Adiposity in Obesity-Prone Carbohydrate-Sensitive Rats.

BACKGROUND: We have reported large differences in adiposity (fat mass/body weight) gain between rats fed a low-fat, high-starch diet, leading to their classification into carbohydrate "sensitive" and "resistant" rats. In sensitive animals, fat accumulates in visceral adipose tissues, leading to the suggestion that this form of obesity could be responsible for rapid development of metabolic syndrome. OBJECTIVE: We investigated whether increased amylase secretion by the pancreas and accelerated starch degradation in the intestine could be responsible for this phenotype. METHOD: Thirty-two male Wistar rats (7-wk-old) were fed a purified low-fat (10%), high-carbohydrate diet for 6 wk, in which most of the carbohydrate (64% by energy) was provided as corn starch. Meal tolerance tests of the Starch diet were performed to measure glucose and insulin responses to meal ingestion. Indirect calorimetry combined with use of 13C-labelled dietary starch was used to assess meal-induced changes in whole body and starch-derived glucose oxidation. Real-time polymerase chain reaction was used to assess mRNA expression in pancreas, liver, white and brown adipose tissues, and intestine. Amylase activity was measured in the duodenum, jejunum, and ileum contents. ANOVA and regression analyses were used for statistical comparisons. RESULTS: "Resistant" and "sensitive" rats were separated according to adiposity gain during the study (1.73% ± 0.20% compared with 4.35% ± 0.36%). Breath recovery of 13CO2 from 13C-labelled dietary starch was higher in "sensitive" rats, indicating a larger increase in whole body glucose oxidation and, conversely, a larger decrease in lipid oxidation. Amylase mRNA expression in pancreas, and amylase activity in jejunum, were also higher in sensitive rats. CONCLUSION: Differences in digestion of starch can promote visceral fat accumulation in rats when fed a low-fat, high-starch diet. This mechanism may have important implications in human obesity.

Low-protein and methionine, high-starch diets increase energy intake and expenditure, increase FGF21, decrease IGF-1, and have little effect on adiposity in mice.

Low-protein diets most often induce increased energy intake in an attempt to increase protein intake to meet protein needs with a risk of accumulation as fat of the excess energy intake. In female adult BALB/c mice, a decrease in dietary casein from 20% to 6% and 3% increased energy intake and slightly increased adiposity, and this response was exacerbated with soy proteins with low methionine content. The effect on fat mass was however limited because total energy expenditure increased to the same extent as energy intake. Lean body mass was preserved in all 6% fed mice and reduced only in 3% casein-fed animals. Insulin response to an oral glucose tolerance test was reduced in soy-fed mice and in low-protein-fed mice. Low-protein diets did not affect uncoupling protein 1 and increased fibroblast growth factor 21 (FGF21) in brown adipose tissue and increased FGF21, fatty acid synthase, and cluster of differentiation 36 in the liver. In the hypothalamus, neuropeptide Y was increased and proopiomelanocortin was decreased only in 3% casein-fed mice. In plasma, when protein was decreased, insulin-like growth factor-1 decreased and FGF21 increased and plasma FGF21 was best described by using a combination of dietary protein level, protein-to-carbohydrate ratio, and protein-to-methionine ratio in the diet. In conclusion, reducing dietary protein and protein quality increases energy intake but also energy expenditure resulting in an only slight increase in adiposity. In this process, FGF21 is probably an important signal that responds to a complex combination of protein restriction, protein quality, and carbohydrate content of the diet.

AMPK Activation Reduces Hepatic Lipid Content by Increasing Fat Oxidation In Vivo.

The energy sensor AMP-activated protein kinase (AMPK) is a key player in the control of energy metabolism. AMPK regulates hepatic lipid metabolism through the phosphorylation of its well-recognized downstream target acetyl CoA carboxylase (ACC). Although AMPK activation is proposed to lower hepatic triglyceride (TG) content via the inhibition of ACC to cause inhibition of de novo lipogenesis and stimulation of fatty acid oxidation (FAO), its contribution to the inhibition of FAO in vivo has been recently questioned. We generated a mouse model of AMPK activation specifically in the liver, achieved by expression of a constitutively active AMPK using adenoviral delivery. Indirect calorimetry studies revealed that liver-specific AMPK activation is sufficient to induce a reduction in the respiratory exchange ratio and an increase in FAO rates in vivo. This led to a more rapid metabolic switch from carbohydrate to lipid oxidation during the transition from fed to fasting. Finally, mice with chronic AMPK activation in the liver display high fat oxidation capacity evidenced by increased [C14]-palmitate oxidation and ketone body production leading to reduced hepatic TG content and body adiposity. Our findings suggest a role for hepatic AMPK in the remodeling of lipid metabolism between the liver and adipose tissue.

Low-protein diet-induced hyperphagia and adiposity are modulated through interactions involving thermoregulation, motor activity, and protein quality in mice.

Low protein (LP)-containing diets can induce overeating in rodents and possibly in humans in an effort to meet protein requirement, but the effects on energy expenditure (EE) are unclear. The present study evaluated the changes induced by reducing dietary protein from 20% to 6%-using either soy protein or casein-on energy intake, body composition, and EE in mice housed at 22°C or at 30°C (thermal neutrality). LP feeding increased energy intake and adiposity, more in soy-fed than in casein-fed mice, but also increased EE, thus limiting fat accumulation. The increase in EE was due mainly to an increase in spontaneous motor activity related to EE and not to thermoregulation. However, the high cost of thermoregulation at 22°C and the subsequent heat exchanges between nonshivering thermogenesis, motor activity, and feeding induced large differences in adaptation between mice housed at 22°C and at 30°C.

Fructo-oligosaccharides reduce energy intake but do not affect adiposity in rats fed a low-fat diet but increase energy intake and reduce fat mass in rats fed a high-fat diet.

The ingestion of low or high lipid diets enriched with fructo-oligosaccharide (FOS) affects energy homeostasis. Ingesting protein diets also induces a depression of energy intake and decreases body weight. The goal of this study was to investigate the ability of FOS, combined or not with a high level of protein (P), to affect energy intake and body composition when included in diets containing different levels of lipids (L). We performed two studies of similar design over a period of 5weeks. During the first experiment (exp1), after a 3-week period of adaptation to a normal protein-low fat diet, the rats received one of the following four diets for 5weeks (6 rats per group): (i) normal protein (14% P/E (Energy) low fat (10% L/E) diet, (ii) normal protein, low fat diet supplemented with 10% FOS, (iii) high protein (55%P/E) low fat diet, and (iv) high protein, low fat diet supplemented with 10% FOS. In a second experiment (exp2) after the 3-week period of adaptation to a normal protein-high fat diet, the rats received one of the following 4 diets for 5weeks (6 rats per group): (i) normal protein, high fat diet (35% of fat), (ii) normal protein, high fat diet supplemented with 10% FOS, (iii) high protein high fat diet and (iv) high protein high fat diet supplemented with 10% FOS. In low-fat fed rats, FOS did not affect lean body mass (LBM) and fat mass but the protein level reduced fat mass and tended to reduce adiposity. In high-fat fed rats, FOS did not affect LBM but reduced fat mass and adiposity. No additive or antagonistic effects between FOS and the protein level were observed. FOS reduced energy intake in low-fat fed rats, did not affect energy intake in normal-protein high-fat fed rats but surprisingly, and significantly, increased energy intake in high-protein high-fat fed rats. The results thus showed that FOS added to a high-fat diet reduced body fat and body adiposity.

Modifying the Dietary Carbohydrate-to-Protein Ratio Alters the Postprandial Macronutrient Oxidation Pattern in Liver of AMPK-Deficient Mice.

Background: Hepatic AMP-activated kinase (AMPK) activity is sensitive to the dietary carbohydrate-to-protein ratio. However, the role of AMPK in metabolic adaptations to variations in dietary macronutrients remains poorly understood.Objective: The objective of this study was to determine the role of hepatic AMPK in the adaptation of energy metabolism in response to modulation of the dietary carbohydrate-to-protein ratio.Methods: Male 7-wk-old wild-type (WT) and liver AMPK-deficient (knockout) mice were fed either a normal-protein and normal-carbohydrate diet (NP-NC; 14% protein, 76% carbohydrate on an energy basis), a low-protein and high-carbohydrate diet (LP-HC; 5% protein, 85% carbohydrate), or a high-protein and low-carbohydrate diet (HP-LC; 55% protein, 35% carbohydrate) for 3 wk. During this period, after an overnight fast, metabolic parameters were measured and indirect calorimetry was performed in mice during the first hours after refeeding a 1-g calibrated meal of their own diet in order to investigate lipid and carbohydrate metabolism.Results: Knockout mice fed an LP-HC or HP-LC meal exhibited 24% and 8% lower amplitudes in meal-induced carbohydrate and lipid oxidation changes. By contrast, knockout mice fed an NP-NC meal displayed normal carbohydrate and lipid oxidation profiles. These mice exhibited a transient increase in hepatic triglycerides and a decrease in hepatic glycogen. These changes were associated with a 650% higher secretion of fibroblast growth factor 21 (FGF21) 2 h after refeeding.Conclusions: The consequences of hepatic AMPK deletion depend on the dietary carbohydrate-to-protein ratio. In mice fed the NP-NC diet, deletion of AMPK in the liver led to an adaptation of liver metabolism resulting in increased secretion of FGF21. These changes possibly compensated for the absence of hepatic AMPK, as these mice exhibited normal postprandial changes in carbohydrate and lipid oxidation. By contrast, in mice fed the LP-HC and HP-LC diets, the lack of adjustment in liver metabolism in knockout mice resulted in a metabolic inflexibility, leading to a reduced amplitude of meal-induced changes in carbohydrate and lipid oxidation.

Metabolic effects of intermittent access to caloric or non-caloric sweetened solutions in mice fed a high-caloric diet.

Human consumption of obesogenic diets and soft drinks, sweetened with different molecules, is increasing worldwide, and increases the risk of metabolic diseases. We hypothesized that the chronic consumption of caloric (sucrose, high-fructose corn syrup (HFCS), maltodextrin) and non-caloric (sucralose) solutions under 2-hour intermittent access, alongside the consumption of a high-fat high-sucrose diet, would result in differential obesity-associated metabolic abnormalities in mice. Male C57BL/6 mice had ad libitum access to an HFHS diet and to water (water control group). In addition, some mice had access, 2h/day, 5days/week (randomly chosen) for 12weeks, to different solutions: i) a sucrose solution (2.1kJ/ml), ii) an HFCS solution (2.1kJ/ml), iii) a maltodextrin solution (2.1kJ/ml) and a sucralose solution (60mM) (n=15/group). Despite no changes in total caloric intake, 2h-intermittent access to the sucrose, HFCS or maltodextrin solutions led to increased body weight and accumulation of lipids in the liver when compared to the group consuming water only. The HFCS and sucrose solutions induced a higher fat mass in various fat depots, glucose intolerance, increased glucose oxidation at the expense of lipid oxidation, and a lower hypothalamic expression of NPY in the fasted state. HFCS also reduced proopiomelanocortin expression in the hypothalamus. 2h-intermittent access to sucralose did not result in significant changes in body composition, but caused a stronger expression of CART in the hypothalamus. Finally, sucrose intake showed a trend to increase the expression of various receptors in the nucleus accumbens, linked to dopamine, opioid and endocannabinoid signaling. In conclusion, 2h-intermittent access to caloric solutions (especially those sweetened with sucrose and HFCS), but not sucralose, resulted in adverse metabolic consequences in high-fat high-sucrose-fed mice.

Increased Cost of Motor Activity and Heat Transfer between Non-Shivering Thermogenesis, Motor Activity, and Thermic Effect of Feeding in Mice Housed at Room Temperature - Implications in Pre-Clinical Studies.

The components of energy expenditure, total metabolic rate (TMR), resting metabolic rate (RMR), thermogenic response to feeding (TEF), activity, and cost of activity were measured in fed and fasted mice housed at 22 and 30°C. Mice housed at 22°C had more than two times larger TMR and RMR. Mice at 22°C were less active when fasted but more active when fed. Cost of activity was nearly doubled in the fasted and in the fed state. Analysis of the short-term relation between TMR, RMR, and bouts of activity showed that, at 22°C, the bouts of activity induced a decrease in the intensity of RMR that reflected the reduced need for thermal regulation induced by the heat released from muscular contraction. This phenomenon induced a considerable underestimation of TEF and prevented its reliable measurement when mice were housed at 22°C. Correlation between TMR and activity measured across time in individual mice was very strong at both 22 and 30°C, but the correlation measured across mice was much weaker at 30°C and no longer significant at 22°C. We suspect that this phenomenon was due to the fact that RMR is a much more reliable predictor of TMR than activity. RMR is more variable at 22°C than at 30°C because of heat transfers between thermal regulation and heat released by other discontinuous processes, such as activity and TEF. Therefore, more noise is introduced into the correlations performed across multiple mice between TMR and activity at 22°C. On the other hand, it should be kept in mind that the doubling of TMR and RMR at 22°C is fueled by an increased non-shivering thermogenesis that can obviously modify how the mouse responds to pharmacological and nutritional challenges. Taken together, these results suggest that in pre-clinical studies, mice should be housed in conditions where thermal regulation is limited as is generally the case in humans. However, the increased sensitivity of mice to small changes in ambient temperature can also be used as a versatile tool to investigate the role of thermal regulation on the energy balance equation in humans.