Protein degradation and peptide release from milk proteins in human jejunum. Comparison with in vitro gastrointestinal simulation.

Human jejunal digests after oral ingestion of casein and whey protein were collected by a nasogastric tube and protein degradation and peptide release was compared with that found in the digests of the same substrates using a standardised protocol. No intact casein was detected in the jejunal nor in the in vitro samples taken during the intestinal phase, while ß-lactoglobulin was found in one hour-jejunal samples in agreement with the in vitro digestion. In vivo and in vitro digests showed comparable peptide profiles and high number of common sequences. A selective precipitation step was used to strengthen the identification of phosphorylated peptides. Most of the sequences found in jejunum, some of them not previously described, were also identified in the simulated digests. Common resistant regions to digestion were identified, revealing that the in vitro protocol constitutes a good approximation to the physiological gastrointestinal digestion of milk proteins.

Combination of micellar casein with calcium and vitamins D2 and K2 improves bone status of ovariectomized mice.

UNLABELLED: Nutritional approaches may help to preserve bone quality. The purpose of our study was to demonstrate the efficiency of an innovative bone health product (BHP) including micellar casein rich in calcium, vitamin D2 and vitamin K2, to improve bone mineral density. INTRODUCTION: The aim of postmenopausal osteoporosis treatment is to decrease bone resorption and/or increase bone formation. Because of the slow bone turnover, osteoporosis prevention and therapies are long-lasting, implying great costs and poor compliance. Even if the effects of nutrition on bone are not as marked as that of pharmaceutical agents, it can be of great help. The purpose of our study was to demonstrate the efficiency of an innovative bone health product (BHP) containing micellar casein rich in calcium, vitamin D2 and vitamin K2, for the improvement of bone mineral density (BMD). METHODS: An ovariectomized mice model was used to study the effect of different concentrations of the ingredient on BMD and microarchitectural parameters. Blood concentrations of C-terminal telopeptide of type I collagen (CTX), N-terminal propeptide of type 1 procollagene (PINP), alkaline phosphatase (ALP), osteocalcin (OC) and RANKL were also measured to evaluate bone remodelling, To evaluate the efficiency of the product to modulate osteoblast and osteoclast growth and differentiation, primary murine bone cells were used. RESULTS: In vivo studies showed that BMD and microarchitectural parameters were dose-dependently improved after ingestion of the supplement for 3 months. We also report increased osteoblast activity as shown by increased OC activity and decreased osteoclastogenesis as shown by reduced CTX activity. In vitro studies support that BHPs stimulate osteoblast differentiation and mineralization and inhibit osteoclast resorption activity. CONCLUSION: Our results show that, when chronically ingested, BHPs improve BMD of ovariectomized mice. This work supports that providing an ingredient including micellar casein rich in calcium, vitamin D2 and vitamin K2 is more efficient than the control diet to maintain bone quality.

Environmental enrichment and cafeteria diet attenuate the response to chronic variable stress in rats.

Exposure to an enriched environment (EE) or the intake of a highly palatable diet may reduce the response to chronic stress in rodents. To further explore the relationships between EE, dietary intake and stress, male Sprague-Dawley rats were fed one of two diets for 5 weeks: high carbohydrate (HC) or "cafeteria" (CAF) (Standard HC plus a choice of highly palatable cafeteria foods: chocolate, biscuits, and peanut butter). In addition, they were either housed in empty cages or cages with EE. After the first two weeks, half of the animals from each group were stressed daily using a chronic variable stress (CVS) paradigm, while the other half were kept undisturbed. Rats were sacrificed at the end of the 5-week period. The effects of stress, enrichment and dietary intake on animal adiposity, serum lipids, and stress hormones were analyzed. Results showed an increase in intra-abdominal fat associated with the CAF diet and an increase in body weight gain associated with both the CAF diet and EE. Furthermore, the increase in ACTH associated with CVS was attenuated in the presence of EE and the CAF diet independently while the stress-induced increase in corticosterone was reduced by the combination of EE and CAF feeding. The present study provides evidence that the availability of a positive environment combined to a highly palatable diet increases resilience to the effects of CVS in rats. These results highlight the important place of palatable food and supportive environments in reducing central stress responses.

High dietary protein intake, reducing or eliciting insulin resistance?

Dietary proteins have an insulinotropic effect and thus promote insulin secretion, which indeed leads to enhanced glucose clearance from the blood. In the long term, however, a high dietary protein intake is associated with an increased risk of type 2 diabetes. Moreover, branched-chain amino acids (BCAA), a prominent group of amino acids, were recently identified to be associated with diabetes. Observational data and intervention studies do not point in the same direction regarding the effect of protein intake on insulin sensitivity and diabetes risk. Therefore, the first aim of this review will be to discuss human studies addressing high dietary protein intake and insulin action, with special attention for BCAA. In the second part, we will highlight the (patho) physiological consequences of high-protein diets regarding insulin action, in particular the role of the mechanistic target of the rapamycin pathway.

Current issues in determining dietary protein and amino-acid requirements.

Pregnancy and the first two years of life are periods of rapid growth and yet the knowledge of requirements for protein and dietary indispensable amino acids is very limited. The development of carbon oxidation methods opens the way to studies that should fill these important gaps in knowledge.

Endogenous proteins in the ileal digesta of adult humans given casein-, enzyme-hydrolyzed casein- or crystalline amino-acid-based diets in an acute feeding study.

BACKGROUND/OBJECTIVES: To ascertain if the form of dietary nitrogen (free amino acids (AA), small peptides, or intact protein) affects the endogenous nitrogen containing substances lost from the upper digestive tract of humans. SUBJECTS/METHODS: Digesta were collected via a naso-ileal tube from the terminal ileum of 16 adult humans in a single parallel study following an acute feeding regimen. Subjects were given an iso-nitrogenous and isocaloric test meal containing 150 g of casein (CAS) (n=6), enzyme-hydrolyzed casein (HCAS) (n=5) or crystalline AA (n=5) dissolved in 550 ml of water, as the sole sources of nitrogen. RESULTS: The mean concentrations and flows of total nitrogen, protein nitrogen, and soluble protein nitrogen passing the terminal ileum were significantly higher (P <0.01) for the CAS and HCAS test-meal groups compared to the AA meal group. Dietary CAS and HCAS had a considerable influence on digesta mucin concentrations and flows compared to free AA (+41%). Only 3-4% of the total nitrogen remained unidentified. CONCLUSIONS: The form of dietary nitrogen (protein, small peptides or free AA) had an acute effect upon the secretion or reabsorption of endogenous proteins in the small intestine of healthy humans, as evident from significant differences in both the quantity and composition of the proteins found in digesta at the end of the ileum.

IKKß in intestinal epithelial cells regulates allergen-specific IgA and allergic inflammation at distant mucosal sites.

Regulation of allergic responses by intestinal epithelial cells (IECs) remains poorly understood. Using a model of oral allergen sensitization in the presence of cholera toxin as adjuvant and mice with cell-specific deletion of inhibitor-κB kinase (IKKß) in IECs (IKKß(ΔIEC)), we addressed the contribution of IECs to allergic sensitization to ingested antigens and allergic manifestations at distant mucosal site of the airways. Cholera toxin induced higher pro-inflammatory responses and altered the profile of the gut microbiota in IKKß(ΔIEC) mice. Antigen-specific immunoglobulin E (IgE) responses were unaltered in IKKß(ΔIEC) mice, but their IgA antibodies (Abs), T helper type 1 (Th1) and Th17 responses were enhanced. Upon nasal antigen challenge, these mice developed lower levels of allergic lung inflammation, which correlated with higher levels of IgA Abs in the airways. The IKKß(ΔIEC) mice also recruited a higher number of gut-sensitized T cells in the airways after nasal antigen challenge and developed airway hyper-responsiveness, which were suppressed by treatment with anti-interleukin-17A. Fecal microbiota transplant during allergic sensitization reduced Th17 responses in IKKß(ΔIEC) mice, but did not affect IgA Ab responses. In summary, we show that IKKß in IECs shapes the gut microbiota and immune responses to ingested antigens and influences allergic responses in the airways via regulation of IgA Ab responses.

Control of protein and energy intake - brain mechanisms.

The protein content of the diet has long been investigated for its influence on food behavior. High-protein diets promote satiety and reduce calorie intake, whereas results for low-protein diets are more contradictory and less established. Protein sensing might take place in the oral cavity or in the post-oral gastrointestinal tract, where specific receptors have been found. Protein signaling to the brain may act through the vagal nerve and involve gastric hormones, such as cholecystokinin and peptide YY. Other pathways are post-absorptive signaling and the direct influence of brain levels of amino acids. High-protein diet enhances the activity of brain satiety centers, mainly the nucleus of the solitary tract and arcuate nucleus, although the activity of brain reward centers might also be modified. A better understanding of the role of both homeostatic and hedonic systems is needed to fully describe the influence of protein on food intake.

Energy restriction only slightly influences protein metabolism in obese rats, whatever the level of protein and its source in the diet.

BACKGROUND: High protein (HP) diets during energy restriction have been studied extensively regarding their ability to reduce body fat and preserve lean body mass, but little is known about their effects on protein metabolism in lean tissues. OBJECTIVE: To determine the effects of energy restriction and protein intake on protein anabolism and catabolism in rats. METHODS: For 5 weeks, 56 male Wistar rats were fed an obesity induction (OI) diet . They were then subjected to a 40% energy restriction using the OI diet or a balanced HP diet for 3 weeks, whereas a control group was fed the OI diet ad libitum (n=8 per group). HP-restricted rats were divided into five groups differing only in terms of their protein source: total milk proteins, casein (C), whey (W), a mix of 50% C and W, and soy (n=8). The animals were then killed in the postprandial state and their body composition was determined. Protein synthesis rates were determined in the liver, gastrocnemius and kidney using a subcutaneous (13)C valine flooding dose. mRNA levels were measured for key enzymes involved in the three proteolysis pathways. RESULTS: Energy restriction, but not diet composition, impacted weight loss and adiposity, whereas lean tissue mass (except in the kidney) was not influenced by diet composition. Levels of neoglucogenic amino acids tended to fall under energy restriction (P<0.06) but this was reversed by a high level of protein. The postprandial protein synthesis rates in different organs were similar in all groups. By contrast, mRNA levels encoding proteolytic enzymes rose under energy restriction in the muscle and kidney, but this was counteracted by a HP level. CONCLUSIONS: In adult obese rats, energy restriction but not diet composition affected fat pads and had little impact on protein metabolism, despite marked effects on proteolysis in the kidney and muscle.

A cafeteria diet modifies the response to chronic variable stress in rats.

Stress is known to lead to metabolic and behavioral changes. To study the possible relationships between stress and dietary intake, male Sprague-Dawley rats were fed one of three diets for 6 weeks: high carbohydrate (HC), high fat (HF), or "Cafeteria" (CAF) (Standard HC plus a choice of highly palatable cafeteria foods: chocolate, biscuits, and peanut butter). After the first 3 weeks, half of the animals from each group (experimental groups) were stressed daily using a chronic variable stress (CVS) paradigm, while the other half of the animals (control groups) were kept undisturbed. Rats were sacrificed at the end of the 6-week period. The effects of stress and dietary intake on animal adiposity, serum lipids, and corticosterone were analyzed. Results showed that both chronic stress and CAF diet resulted in elevated total cholesterol, increased low-density lipoprotein (LDL), and lower high-density lipoprotein (HDL). In addition, increases in body weight, food intake, and intra-abdominal fat were observed in the CAF group compared with the other dietary groups. In addition, there was a significant interaction between stress and diet on serum corticosterone levels, which manifest as an increase in corticosterone levels in stressed rats relative to non-stressed controls in the HC and HF groups but not in the CAF group. These results show that a highly palatable diet, offering a choice of food items, is associated with a reduction in the response to CVS and could validate a stressor-induced preference for comfort food that in turn could increase body weight.

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.

Protein intake, calcium balance and health consequences.

High-protein (HP) diets exert a hypercalciuric effect at constant levels of calcium intake, even though the effect may depend on the nature of the dietary protein. Lower urinary pH is also consistently observed for subjects consuming HP diets. The combination of these two effects was suspected to be associated with a dietary environment favorable for demineralization of the skeleton. However, increased calcium excretion due to HP diet does not seem to be linked to impaired calcium balance. In contrast, some data indicate that HP intakes induce an increase of intestinal calcium absorption. Moreover, no clinical data support the hypothesis of a detrimental effect of HP diet on bone health, except in a context of inadequate calcium supply. In addition, HP intake promotes bone growth and retards bone loss and low-protein diet is associated with higher risk of hip fractures. The increase of acid and calcium excretion due to HP diet is also accused of constituting a favorable environment for kidney stones and renal diseases. However, in healthy subjects, no damaging effect of HP diets on kidney has been found in either observational or interventional studies and it seems that HP diets might be deleterious only in patients with preexisting metabolic renal dysfunction. Thus, HP diet does not seem to lead to calcium bone loss, and the role of protein seems to be complex and probably dependent on other dietary factors and the presence of other nutrients in the diet.

Inhibition of food intake induced by acute stress in rats is due to satiation effects.

Acute mild stress induces an inhibition of food intake in rats. In most studies, the cumulative daily food intake is measured but this only provides a quantitative assessment of ingestive behavior. The present study was designed to analyze the reduction in food intake induced by acute stress and to understand which behavioral and central mechanisms are responsible for it. Two different stressors, restraint stress (RS) and forced swimming stress (FSS), were applied acutely to male Wistar rats. We first measured corticosterone and ACTH in plasma samples collected immediately after acute RS and FSS in order to validate our stress models. We measured food intake after RS and FSS and determined meal patterns and behavioral satiety sequences. The expressions of CRF, NPY and POMC in the hypothalamus were also determined immediately after acute RS and FSS. The rise in corticosterone and ACTH levels after both acute RS and FSS validated our models. Furthermore, we showed that acute stress induced a reduction in cumulative food intake which lasted the whole day for RS but only for the first hour after FSS. For both stressors, this stress-induced food intake inhibition was explained by a decrease in meal size and duration, but there was no difference in ingestion speed. The behavioral satiety sequence was preserved after RS and FSS but grooming was markedly increased, which thus competed with, and could reduce, other behaviors, including eating. Lastly, we showed that RS induced an increase in hypothalamic POMC expression. These results suggest that acute stress may affect ingestive behavior by increasing satiation and to some extent by enhancing grooming, and this may be due to stimulation of the hypothalamic POMC neurons.

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.

Dietary protein, weight loss, and weight maintenance.

The role of dietary protein in weight loss and weight maintenance encompasses influences on crucial targets for body weight regulation, namely satiety, thermogenesis, energy efficiency, and body composition. Protein-induced satiety may be mainly due to oxidation of amino acids fed in excess, especially in diets with "incomplete" proteins. Protein-induced energy expenditure may be due to protein and urea synthesis and to gluconeogenesis; "complete" proteins having all essential amino acids show larger increases in energy expenditure than do lower-quality proteins. With respect to adverse effects, no protein-induced effects are observed on net bone balance or on calcium balance in young adults and elderly persons. Dietary protein even increases bone mineral mass and reduces incidence of osteoporotic fracture. During weight loss, nitrogen intake positively affects calcium balance and consequent preservation of bone mineral content. Sulphur-containing amino acids cause a blood pressure-raising effect by loss of nephron mass. Subjects with obesity, metabolic syndrome, and type 2 diabetes are particularly susceptible groups. This review provides an overview of how sustaining absolute protein intake affects metabolic targets for weight loss and weight maintenance during negative energy balance, i.e., sustaining satiety and energy expenditure and sparing fat-free mass, resulting in energy inefficiency. However, the long-term relationship between net protein synthesis and sparing fat-free mass remains to be elucidated.

Adaptation of lipid-induced satiation is not dependent on caloric density in rats.

UNLABELLED: Food intake is modulated by ingestive (gastrointestinal) and post-ingestive signals; ingested fat is potent to produce short-term satiety (satiation) but this can be modified by long-term ingestion of a high fat diet. AIM: Determine whether altered lipid-induced satiation is dependent on the fat content of the diet, rather than increased caloric density or changes in adiposity. METHODS: Initial experiments determined the differences in the microstructure of meal patterns in rats fed a high fat diet (HF: 38% fat kcal) and in rats pair-fed an isocaloric, isonitrogenous low fat diet (LF: 10% fat kcal) and changes in meal patterns measured after long-term maintenance on the HF diet. RESULTS: Rats fed the HF diet had a significant 50% increase in meal frequency compared to rats fed the LF diet; in addition, there was a significant reduction in meal size (32%) and inter meal interval (38%) consistent with induction of satiation. After 8 weeks on the HF diet, these parameters tend to approach those of rats maintained on the LF diet. There was a significant 56% decrease in the activation of neurons in the NTS in response to intragastric gavage of lipid in rats maintained for 8 weeks on the HF compared to LF diet. CONCLUSION: Dietary fat alters meal patterns consistent with induction of a short-term satiety signal. This signal is attenuated with long-term exposure to dietary lipid, in the absence of ingestion of additional calories or changes in body weight. This adaptation of short-term satiety might contribute to diet-induced obesity.

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..

Effects of amino acid-derived luminal metabolites on the colonic epithelium and physiopathological consequences.

Depending on the amount of alimentary proteins, between 6 and 18 g nitrogenous material per day enter the large intestine lumen through the ileocaecal junction. This material is used as substrates by the flora resulting eventually in the presence of a complex mixture of metabolites including ammonia, hydrogen sulfide, short and branched-chain fatty acids, amines; phenolic, indolic and N-nitroso compounds. The beneficial versus deleterious effects of these compounds on the colonic epithelium depend on parameters such as their luminal concentrations, the duration of the colonic stasis, the detoxication capacity of epithelial cells in response to increase of metabolite concentrations, the cellular metabolic utilization of these metabolites as well as their effects on colonocyte intermediary and oxidative metabolism. Furthermore, the effects of metabolites on electrolyte movements through the colonic epithelium must as well be taken into consideration for such an evaluation. The situation is further complicated by the fact that other non-nitrogenous compounds are believed to interfere with these various phenomenons. Finally, the pathological consequences of the presence of excessive concentrations of these compounds are related to the short- and, most important, long-term effects of these compounds on the rapid colonic epithelium renewing and homeostasis.

Commercial Phaseolus vulgaris extract (starch stopper) increases ileal endogenous amino acid and crude protein losses in the growing rat.

The effect of a commercial Phaseolus vulgaris extract (PVE, starch stopper) on ileal and fecal endogenous protein losses was studied. Growing rats were fed for 14 days a protein-free diet containing PVE at a nutritional concentration of 0% (PF1), 0.4% (PF2), or 1.1% PVE (PF3) or 1.1% autoclaved PVE (PF4). An indigestible marker (TiO(2)) was included in each diet. Ileal endogenous amino acid (AA) losses were significantly higher (P < 0.05) in PF3 (20% higher than in PF1), except for Pro, Gly, Ala, and His. Endogenous ileal N losses were 22% higher in PF3 than in PF1. Endogenous fecal AA and N losses were all significantly higher (P < 0.05) in PF3. Starch digestibility ( approximately 100%), food intake (single daily meal, d10-23), and body weight loss were not significantly different among the groups. PVE, at 1.1% of the diet, not only was ineffective in reducing starch digestibility but also led to increased ileal endogenous N losses, possibly due to the antinutritional factors (trypsin inhibitor, lectin) present in the PVE.