Hydrolyzed collagen improves bone status and prevents bone loss in ovariectomized C3H/HeN mice.

SUMMARY: This study evaluates the effect of hydrolyzed collagen (HC) on bone health of ovariectomized mice (OVX) at different ages. Twenty-six weeks after the OVX procedure, HC ingestion was still able to improve significantly bone mineral density (BMD) and some femur biomechanical parameters. Moreover, HC ingestion for 1 month before surgery prevented BMD decrease. INTRODUCTION: HC can play an important role in preserving BMD before osteoporosis appears. The aim of this study was to evaluate the effect of HC on bone health of ovariectomized mice at different ages. METHODS: Female C3H mice were either OVX at 3 or 6 months and fed for 6 months (first experiment) or 3 months (second experiment) with diet including 0, 10, or 25 g/kg of HC. In the second experiment, one group received HC 1 month before surgery, and two groups received the supplementation immediately after surgery, one fed ad libitum and the other by gavage. Mice treated with raloxifene were used as a positive control. BMD, femur intrinsic and extrinsic biomechanical properties, and type I collagen C-terminal telopeptide were measured after 12 and 26 weeks. Food intake and spontaneous physical activity were also recorded. RESULTS: The OVX procedure increased body weight, while food intake decreased, thus suggesting that resting metabolism was decreased. Ingestion of 25 g/kg of HC for 3 or 6 months reduced bone loss significantly in, respectively, 3- and 6-month-old OVX mice. The lowest HC concentration was less efficient. HC ingestion for 3 months is as efficient as raloxifene to protect 3-month-old OVX mice from bone loss. Our results also demonstrated that HC ingestion before surgery prevented the BMD decreases. CONCLUSION: This study confirms that dietary collagen reduces bone loss in OVX mice by increasing the diameter of the cortical areas of femurs and can have a preventive effect.

Peripherally injected cholecystokinin-induced neuronal activation is modified by dietary composition in mice.

The aim of this study was to investigate the effect of long-term nutrient intake on the central response to the anorexigenic gut hormone CCK. C57BL/6 mice were fed one of three diets for 6 weeks: standard high carbohydrate (HC), high fat (HF), or high protein (HP). Assessment of brain response to cholecystokinin (CCK) by manganese-enhanced MRI (MEMRI) showed a reduction in neuronal activity both in an appetite-related area (ventromedial nucleus of the hypothalamus) and areas associated with reward (nucleus accumbens and striatum) regardless of diet. When comparing diet effects, while the HF diet did not induce any change in activity, reductions in MEMRI-associated signal were found in the paraventricular nucleus (PVN) and lateral hypothalamic area (LHA) when comparing the HP to the HC diet. In addition, a significant interaction was found between CCK administration and the HF diet, shown by an increased activation in the PVN, which suggests a decrease the inhibiting action of CCK. Our results put forward that the long-term intake of an HP diet leads to a reduction in basal hypothalamic activation while a high-fat diet leads to desensitization to CCK-induced effects in the hypothalamus.

Alterations of lipid metabolism and gene expression in rat adipocytes during chronic olanzapine treatment.

Although antipsychotics are established drugs in schizophrenia treatment, they are admittedly known to induce side effects favoring the onset of obesity and worsening its complications. Despite potential involvement of histamine receptor antagonism, or of other neurotransmitter systems, the mechanism by which antipsychotic drugs increase body weight is not elucidated. The aim of the present study was to investigate whether chronic antipsychotic treatments can directly alter the regulation of two main functions of white adipose tissue: lipolysis and glucose utilization. The influence of a classical antipsychotic (haloperidol) was compared to that of two atypical antipsychotics, one known to favor weight gain (olanzapine), the other not (ziprasidone). Cell size, lipolytic capacity and glucose transport activity were determined in white adipocytes of rats subjected to 5-week oral treatment with these antipsychotics. Gene expression of adipocyte proteins involved in glucose transport or fat storage and mobilization, such as glucose transporters (GLUT1 and GLUT4), leptin, matrix metallo-proteinase-9 (MMP9), hormone-sensitive lipase (HSL) and fatty acid synthase (FAS) was also evaluated. Adipocytes from chronic olanzapine-treated rats exhibited decreased lipolytic activity, lowered HSL expression and increased FAS expression. These changes were concomitant to enlarged fat deposition and adipocyte size. Alterations were observed in adipocytes from olanzapine-treated rats whereas the other antipsychotics did not induce any notable disorder. Our results therefore show evidence of an effect of chronic antipsychotic treatment on rat adipocyte metabolism. Thus, impairment of fat cell lipolysis should be considered as a side effect of certain antipsychotics, leading, along with the already documented hyperphagia, to the excessive weight gain observed in patients under prolonged treatment..

A tryptophan-rich protein diet efficiently restores sleep after food deprivation in the rat.

Sleep depends on the quantity and quality of the diet. Several studies have shown that food deprivation results in a reduction in sleep duration. It has also been demonstrated that in the newborn, the supply of certain essential amino acids improves sleep through their action on the synthesis of specific neurotransmitters. The aim of the present study was to test if the quantity and/or quality of dietary protein could improve the recovery of sleep during re-feeding after caloric deprivation. Sleep parameters were compared in rats fed ad libitum, food restricted during 4 days, or reefed isocalorically after food restriction with three dietary regimens varying in terms of the amount (14% versus 30%) or quality (milk protein or alpha-lactalbumin) of protein. The results showed that sleep recovery, in particular slow-wave sleep, was improved in rats re-fed with alpha-lactalbumin. This result confirms the close relationship between feeding and sleep and suggest that alpha-lactabumin could be used to improve sleep in adult submitted to nutritional disturbances such as food restriction, shift work, Ramadan.

Fat-depleted CLA-treated mice enter torpor after a short period of fasting.

Resting energy expenditure (Resting-EE), EE with treadmill exercise, and post-prandial thermogenesis were continuously monitored by indirect calorimetry during a 24 h recording session in control (CT) and CLA-treated (CLA) (1% CLA in the food, by weight) C57Bl/6 male mice. After 15 days of CLA treatment, the fat content of CLA mice had fallen to 20% of that in CT mice. CLA mice were able to face the energy challenge of exercise but used less lipid than CT mice. Resting-EE values fell during the post-exercise period. The thermogenic response to a calibrated test meal given 5 h after the run abolished the differences in EE and substrate oxidation between CT and CLA mice. However, 2.5 h after ingestion of the test meal onward, CT mice gradually increased their lipid oxidation to sustain resting-EE levels. In contrast, CLA mice did not increase their lipid oxidation and their resting-EE levels fell significantly until they entered into torpor. Blood leptin was low but similar in CT and CLA-treated mice suggesting that leptin is not critical to induce torpor. We suggest that the durable inhibition of lipid oxidation in fasting CLA mice was an adaptive behaviour devoted at sparing the residual adipose deposits.

Feeding patterns and meal microstructure during development of a taste aversion to a threonine devoid diet.

Food intake decreases and a conditioned taste aversion is induced when rats are fed a diet that is devoid of an indispensable amino acid. The purpose of this study was to characterize the meal patterns associated with (1) the onset of anorexia after the initial recognition of a threonine deficiency and (2) after the development of the conditioned taste aversion to this deficient diet. When rats ate the threonine-devoid diet for the first time, meal patterns were characterized by an increase in intermeal interval (IMI) between 3 and 6 h after food presentation, which was followed by a decrease in meal size and ingestion rate, between 6 and 12 h. Meal patterns on days 2 and 10 were associated with expression of the taste aversion, characterized by meals of smaller size, longer duration and by a reduction in ingestion rate, without variations in either IMI or meal frequency. Meals of the threonine-deficient group were composed of more frequent bouts, smaller size and shorter duration, with large within-meal pauses, which accounted for the reduced ingestion rate. This study presents the first analysis in terms of feeding patterns and meal microstructure of a conditioned taste aversion induced by a food rather than a toxin.

Prediction of basal metabolism from organ size in the rat: relationship to strain, feeding, age, and obesity.

Use of the weight of various organs and tissues together with their specific metabolic activity for prediction of basal metabolism (BM) seems to be promising. In this study we compared the use of this method with those based on simple or multiple regression analyses. We observed that 97.4% of differences in BM in a group of nine adult male Wistar rats weighing 273--517 g could be accounted for by changes in tissue and organ weights. BM measured in lean Zucker and Sprague-Dawley rats did not diverge from the prediction of the model by >1.6%. According to the organ-based model as well as multiple regression analyses, but not simple regression analyses, BM was increased 18--21% in young rats, decreased 6--7% in food restricted/refed rats, and decreased 19--21% in aged rats. Only with obese rats did the predictions of the two methods diverge. The main reason for this discrepancy seems to be the way adipose tissue size and metabolism are taken into account.

A high-protein meal exceeds anabolic and catabolic capacities in rats adapted to a normal protein diet.

The postprandial fixation of dietary nitrogen in splanchnic and peripheral tissues as well as its dynamic transfer to the nitrogen pools of the body were quantified in rats subjected to an acute augmentation of dietary protein. For this purpose, we traced the dietary protein and studied the immediate fate of exogenous nitrogen in many tissues and biological fluids. Rats were adapted to a diet providing an adequate protein level (14 g/100 g), and then fed a meal containing either 0.42 g (Group A) or 1.50 g (Group H) of [(15)N]-labeled milk protein. The amounts of exogenous nitrogen transferred to urea (0.32 +/- 0.04 vs. 2.46 +/- 0.25 mmol, respectively), incorporated in splanchnic (0.41 +/- 0.02 vs. 0.87 +/- 0.10 mmol) and peripheral (1.65 +/- 0.84 vs. 2.36 +/- 0.49 mmol) tissue protein were higher in group H than in group A. Individual plasma amino acids (AA) [(15)N]-enrichments showed that AA respond differentially to an acute augmentation of dietary intake. This work provides new descriptive and quantitative information on the metabolic fate of dietary nitrogen in the postprandial state. It highlights the higher integration of a surplus of dietary nitrogen in the tissues even if it is rapidly limited by saturation of the protein synthesis capacities. The main metabolic response remains the stimulation of AA degradation, leading to a large rise in urea production. However, both anabolic and catabolic systems are exceeded, resulting in an elevation of peripheral AA and negative feedback on the gastric emptying rate.

Postprandial metabolism and aversive response in rats fed a threonine-devoid diet.

Lack of an indispensable amino acid in the diet induces a rapid reduction in food intake. In this study, we assessed whether the anorectic signal after ingestion of a meal lacking threonine originated from either direct perception of the decrease in plasma threonine or from an indirect effect related to increased postprandial amino acid catabolism and energy expenditure. We observed that 3 g of such a meal was sufficient to induce an aversive response to the diet within 2 h. Postprandial changes to plasma ammonia and urea, urinary urea, and energy metabolism did not differ from those measured after a control meal. In contrast, plasma threonine levels fell within 1 h after the meal. It is concluded that an increase in postprandial energy expenditure is not involved in the anorectic response to eating a threonine-devoid diet. The drop in plasma threonine levels may be a potential signal, but the fact that the decrease in food intake occurred 1 h after the decrease in plasma threonine questions a direct causal relationship.

Hindlimb immobilization applied to 21-day-old mdx mice prevents the occurrence of muscle degeneration.

Dystrophin-deficient skeletal muscles of mdx mice undergo their first rounds of degeneration-regeneration at the age of 14-28 days. This feature is thought to result from an increase in motor activity at weaning. In this study, we hypothesize that if the muscle is prevented from contracting, it will avoid the degenerative changes that normally occur. For this purpose, we developed a procedure of mechanical hindlimb immobilization in 3-wk-old mice to restrain soleus (Sol) and extensor digitorum longus (EDL) muscles in the stretched or shortened position. After a 14-day period of immobilization, the striking feature was the low percentage of regenerated (centronucleated) myofibers in Sol and EDL muscles, regardless of the length at which they were fixed, compared with those on the contralateral side (stretched Sol: 8.4 +/- 6.5 vs. 46.6 +/- 10.3%, P = 0.0008; shortened Sol: 1.2 +/- 1.6 vs. 50.4 +/- 16.4%, P = 0.0008; stretched EDL: 05 +/- 0.5 vs. 32.9 +/- 17.5%, P = 0. 002; shortened EDL: 3.3 +/- 3.1 vs. 34.7 +/- 11.1%, P = 0.002). Total numbers of myofibers did not change with immobilization. This study shows that limb immobilization prevents the occurrence of the first round of myofiber necrosis in mdx mice and suggests that muscle contractions play a role in the skeletal muscle degeneration of dystrophin-deficient mdx mouse muscles.

Rapid fall in plasma threonine followed by increased intermeal interval in response to first ingestion of a threonine-devoid diet in rats.

In most animals, ingestion of a diet lacking an essential amino acid (EAA) gives rise to anorexia within a few hours. The first signal in this feeding response may be the fall in plasma levels of the limiting EAA. In the present study, we measured plasma amino acid levels and food intake after the first exposure to either a threonine-devoid (THR-DEV) or corrected (COR) diet in 16 rats bearing a chronic jugular catheter for blood sampling. Food intake was reduced 165 min (p<0.05) after presentation of the THR-DEV diet. Analysis of the feeding pattern showed that intake was reduced via a four-fold lengthening of the second inter-meal interval. Plasma threonine levels started to fall between 30 and 60 min after onset of the meal (p<0.05). These results, observed in the same rats, lend further support for an early modification of the plasma amino acid pattern in relation to the decrease in feeding of a diet that is EAA deficient.

Substrate oxidation during exercise in the rat cannot fully account for training-induced changes in macronutrients selection.

This study investigated spontaneous dietary adaptation to regular exercise in relation to substrate oxidation measured during exercise. Male Wistar rats were offered permanent access to the three sources of macronutrients supplemented with minerals and vitamins. The rats remained sedentary or were trained daily during 3 weeks at moderate intensity (20 m x min(-1), 2 hours). Body weight, total caloric intake, and macronutrients selection were recorded throughout the experiment. Energy expenditure and substrate oxidation were measured before, during, and after an exercise identical for trained and untrained rats (10 m x min(-1) 1 hour). Training reduced body weight gain (2.27 v 5.57 g x day(-1)), increased protein intake (52.6% v 39.2%), and decreased carbohydrate intake (21.3% v 39.5%). Basal and running energy expenditure, as well as glucose and lipid oxidation, remained essentially comparable in trained and untrained rats. The relative contribution of glucose oxidation (Gox) to total energy expenditure decreased during exercise (52.2%, average of all rats) relative to before exercise (60.8%). Gox during exercise was positively correlated with resting Gox before exercise, showing that preexercise substrate oxidation was a strong determinant of running substrate oxidation. However, the slope was smaller for the trained than for the untrained rats, showing that exercise increases Gox less in trained rats than in untrained ones. We conclude from this study that, since food selection but not substrate oxidation changed following training, food intake adapted to substrate requirements induced by regular training and not the contrary. However, large differences remained between the mixture ingested, in which lipids accounted for only 26% of the energy, and the mixture oxidized during exercise, in which lipids accounted for 50.7% of the substrate oxidized. Such a difference may be related to metabolic requirements during the rest of the day and/or to the distribution of macronutrients intake relative to exercise. This question deserves further investigation with recording of macronutrients selection, energy expenditure, and substrate oxidation over 24 hours.

Metabolic action of neuropeptide Y in relation to its effect on feeding.

Because energy homeostasis depends on a continuous balance between food intake, energy expenditure, and energy storage, it was expected that neuropeptide Y (NPY) could act not only on food intake but also on metabolic parameters. Using an original calorimetric device that allows the computation of the background metabolism (energy expenditure free from the cost of locomotor activity), we assessed the effect of a microinjection of NPY upon the quantitative (background metabolism, thermic effect of food) and qualitative (respiratory quotient) components of energy metabolism. NPY was injected into the juxtafornical hypothalamus at a dose that promotes feeding behavior (1 microg/0.5 microL) and enhances locomotor activity. Although total metabolism was increased proportionally to locomotion, no effect of NPY on background metabolism was observed when no food was available. Only following a calibrated meal given 30 min after the microinjection did NPY induce a delayed decrease in respiratory quotient whereas the postprandial background metabolism remained unaffected. In conclusion, only the new-generation calorimeters can show that the NPY-induced rise in overall metabolic rate is entirely accounted for by the unavoidable enhancement in locomotor activity and that the only metabolic effect of NPY is the delayed postprandial respiratory quotient decrease, suggesting a postabsorptive orientation toward more lipid utilization.

Effect of intraperitoneal injection of glucose on glucose oxidation and energy expenditure in the mdx mouse model of duchenne muscular dystrophy.

Previous studies suggesting that glucose metabolism could be impaired in the skeletal muscles of the mdx mouse led us to study the metabolic response to i.p. injection of either glucose or glucose and insulin in the free-moving mdx mouse. In the first study, changes in blood glucose and plasma insulin levels were measured in mice with chronic venous cannulae. In the second study, the thermogenic response to glucose and the changes induced on glucose oxidation (Gox) and lipid oxidation (Lox) were assessed by indirect calorimetry. The experiments showed that insulin response, as well as whole body glucose uptake, were normal in mdx mice. Addition of exogenous insulin abolished the increase in blood glucose level similarly in mdx and control mice. The thermogenic response to glucose was identical in mdx and control mice but, when insulin was injected with glucose, it increased significantly in mdx mice. Exogenous glucose increased Gox less and decreased Lox less in mdx than in control mice. Addition of exogenous insulin reduced the difference between mdx and control mice but affected Gox and Lox less in mdx than in control mice. Key words Duchenne muscular dystrophy middle dot mdx Mouse middle dot Glucose tolerance test middle dot Respiratory quotient middle dot Energy metabolism middle dot Indirect calorimetry

Components of energy expenditure in the mdx mouse model of Duchenne muscular dystrophy.

Previous observations showing that basal heat production rates and glucose metabolism were reduced in mdx mouse skeletal muscles incubated in vitro led us to study the components of total energy expenditure by open-circuit indirect calorimetry in the intact, free-moving mdx mouse. Our purpose was to verify if the mdx mouse exhibited whole-body alterations in energy metabolism. The results revealed that total and basal energy expenditure, as well as spontaneous activity, energetic cost of activity, and, therefore, energy expended in relation to activity were not significantly different in C57B1/10 (control) and in dystrophic (mdx) mice. In contrast, the thermic effect of food was 32% larger in mdx than in control mice and was accompanied by significant differences in post-prandial glucose and lipid oxidation. The present in vivo study could not show a direct demonstration that impaired glucose metabolism by skeletal muscles participated in this phenomenon. However, since post-prandial glucose metabolism by skeletal muscles contributes a significant part of the thermic effect of food, the present data are in line with previous studies in vitro that show that mdx mouse skeletal muscles probably suffer an impaired control of their energy metabolism.

Effects of treadmill exercise and high-fat feeding on muscle degeneration in mdx mice at the time of weaning.

1. Dystrophin-deficient hindlimb muscles of mdx mice undergo necrosis at the time of weaning when the motor activity of the mice greatly increases and muscle energy metabolism becomes more dependent on insulin and carbohydrates. 2. We have attempted to determine if the onset of myofibre necrosis in mdx mice at the time of weaning is related to the development of motor activity and/or the change in diet. 3. Fourteen-day-old mdx mice were divided into two groups after weaning. One group was trained to run on a treadmill and the other group was kept on a high-fat diet. Muscle necrosis was assessed histologically in the soleus and extensor digitorum longus muscles of mice in both experiments. 4. Keeping mice on a high-fat milk diet from the time of weaning up to 42 days of age did not influence the occurrence of necrosis in the soleus and extensor digitorum longus muscles of the mdx pups. In contrast, treadmill exercise greatly increased necrosis in both muscles. 5. We conclude that an increase in motor activity exacerbates the degeneration of hindlimb muscles of mdx mice at the time of weaning.

Defective regulation of energy metabolism in mdx-mouse skeletal muscles.

Our previous finding of a reduced energy metabolism in slow- and fast-twitch skeletal muscle fibres from the murine model of Duchenne muscular dystrophy (the mdx mouse) led us to examine the importance of intracellular glucose availability for a normal energy turnover. To this end, basal and KCl-stimulated (20.9 mM total extracellular K+) rates of glucose uptake (GUP) and heat production were measured in isolated, glucose-incubated (5 mM) soleus and extensor digitorum longus muscles from mdx and control C57B1/10 mice, in the presence and in the absence of insulin (1.7 nM). Under all conditions and for both muscle types, glucose uptake values for mdx and control muscles were similar although heat production was lower in mdx muscles. The marked stimulation of GUP by insulin in both mdx and control muscles had only minor effects on heat production. In contrast, glucose deprivation or inhibition of glycolysis with 2-deoxy-D-glucose (5 mM) significantly decreased heat production in control muscles only, which attenuated, although did not suppress, the difference in basal heat production between mdx and control muscles. Stimulation of heat production by a short-chain fatty acid salt (octanoate, 2 mM) was significantly less marked in mdx than in control muscles. Increased cytoplasmic synthesis of CoA by addition of 5 mM pantothenate (vitamin B5) increased the thermogenic response to glucose more in mdx than in control muscles. We conclude that the low energy turnover in mdx-mouse muscle fibres is not due to a decrease of intracellular glucose availability, but rather to a decreased oxidative utilization of glucose and free fatty acids. We suggest that some enzyme complex of the tricarboxylic acid cycle or inefficiency of CoA transport in the mitochondria could be involved.

Dystrophin-dependent efficiency of metabolic pathways in mouse skeletal muscles.

Muscles from the mdx mouse (X-linked genetic disorder similar to Duchenne muscular dystrophy) lack dystrophin-associated transsarcolemmal proteins and show reduced maintenance metabolic rates. Here, microcalorimetric comparisons of metabolic stimulation by exogenous substrates in isolated muscles revealed substrate-selective limitation of chemical reaction rates through both glycolytic and TCA-cycle pathways, identical in slow- and fast-twitch mdx muscles. This systemic approach, as opposed to comparisons of single-enzyme activities, sheds new light on the function of dystrophin and associated proteins. The in vivo efficiency of metabolic pathways may depend on stabilization of enzyme complexes by dystrophin-associated elements of the cytoskeleton.

Practical aspects of indirect calorimetry in laboratory animals.

Oxidation of the energetic substrates by the body is associated with oxygen consumption, carbon dioxide production, and heat release specific to the nature of the energetic substrates being oxidized. Therefore, measurement of respiratory exchanges (indirect calorimetry) is a powerful method to investigate heat production of a living organism. In this article, we review the elementary principles of indirect calorimetry and describe the operating principle of the two most typical devices used to perform indirect measurements of energy expenditure in the laboratory animal: the closed-circuit and the open-circuit. We then discuss some practical aspects of the day-to-day use of these devices: respective advantages and limitations of each technique, data processing, calibration, correction for body-size, and computation of the energy expended for activity. In the second part, we review some of the standard formulas of indirect calorimetry that offer the possibility to obtain more precise information such as the rate of oxidation of carbohydrates (CHO), lipids and proteins if some hypotheses are made on the intensity of lipogenic, ketogenic, and gluconeogenic processes. Finally, a practical example of the measurement of energetic cost of activity and thermic effect of food in the rat is given.