N-acetylcysteine dual and antagonistic effect on cadmium cytotoxicity in human leukemia cells.

Although cadmium (Cd2+) is unable to form reactive oxygen species (ROS) directly, many of its adverse effects are connected to increased ROS generation resulting in cell death. In support of this supposition, a large number of studies have shown protective effects of antioxidants such as N-acetylcysteine (NAC) against cadmium induced cytotoxicity. Here, we describe the cytotoxic effects of Cd2+ on human leukemia U937 and K562 cells that were not mediated by oxidative stress. Surprisingly, we observed that addition of low concentrations of NAC can drastically potentiate cadmium cytotoxicity solely via ROS production. However, all adverse effects of the metal were prevented by NAC at high concentrations. Detailed analysis indicated that the protective effect of NAC was mediated by its ability to form stable complex with cadmium [Cd(NAC)2]. In conclusion, NAC exhibits dual and antagonistic effects on Cd2+ cytotoxicity in human leukemia cells.

Mechanical properties of hyaline and repair cartilage studied by nanoindentation.

Articular cartilage is a highly organized tissue that is well adapted to the functional demands in joints but difficult to replicate via tissue engineering or regeneration. Its viscoelastic properties allow cartilage to adapt to both slow and rapid mechanical loading. Several cartilage repair strategies that aim to restore tissue and protect it from further degeneration have been introduced. The key to their success is the quality of the newly formed tissue. In this study, periosteal cells loaded on a scaffold were used to repair large partial-thickness cartilage defects in the knee joint of miniature pigs. The repair cartilage was analyzed 26 weeks after surgery and compared both morphologically and mechanically with healthy hyaline cartilage. Contact stiffness, reduced modulus and hardness as key mechanical properties were examined in vitro by nanoindentation in phosphate-buffered saline at room temperature. In addition, the influence of tissue fixation with paraformaldehyde on the biomechanical properties was investigated. Although the repair process resulted in the formation of a stable fibrocartilaginous tissue, its contact stiffness was lower than that of hyaline cartilage by a factor of 10. Fixation with paraformaldehyde significantly increased the stiffness of cartilaginous tissue by one order of magnitude, and therefore, should not be used when studying biomechanical properties of cartilage. Our study suggests a sensitive method for measuring the contact stiffness of articular cartilage and demonstrates the importance of mechanical analysis for proper evaluation of the success of cartilage repair strategies.

Transient knock-down of kcna2 reduces sleep in larval zebrafish.

In the current study we set out to determine the effects of morpholino oligonucleotide (MO) knock-down of kcna2 on sleep-wake cycles in zebrafish. The results were compared to a non-overlapping MO injection, Dec2, who's mutant is also linked with a short sleep phenotype. Four groups of fish were used in the experiment: naïve fish, and fish injected with either control, kcna2, or Dec2 MO. All groups underwent 24-h behavioral monitoring of sleep-wake cycles at four and seven days-post-fertilization (dpf). First, we established an immobility dependent, sleep related, increase in arousal thresholds at both 4 and 7 dpf. Secondly, we show that kcna2 MO injected fish exhibit significantly less sleep behavior than controls and naïve fish, whereas Dec2 MO injections had similar but less severe effects. Finally, using kcna2 MO injected fish only, we turn to local field recordings at the level of the telencephalon and tectum opticum and rule out that the knock-down resulted in a non-specific increase in neural excitability that would mask sleep behavior.

Modulatory effect of glucose, amino acids, and secretin on CCK-8-induced somatostatin and pancreatic polypeptide release in dogs.

Protein- and fat-rich test meals elicit a strong stimulatory effect on postprandial somatostatin (SLI) and pancreatic polypeptide (PP) release, whereas carbohydrate-rich meals rather attenuate the response of both hormones. Since there is evidence that intestinal hormones might contribute to the postprandial SLI and PP response, it was the aim of the present study to determine in dogs the effect of low-dose cholecystokinin octapeptide (CCK-8) on basal hormone levels and also during a background infusion of amino acids or glucose. In a group of six conscious dogs, sulfated CCK-8 was infused intravenously (i.v.) via a hindleg vein at stepwise increasing infusion rates of 10, 30, and 50 pmol X kg-1 X h. The infusion of CCK was applied during a background infusion of saline (2 ml/min), glucose (0.2 g/min), or an amino acid mixture (8.5%, 2 ml/min). CCK-8 had no effect on plasma insulin and glucagon levels under all experimental conditions. Plasma SLI levels were significantly stimulated by all doses of CCK. This stimulatory effect was similar during background infusions of either saline, glucose, or amino acids, respectively. Pancreatic polypeptide (PP) levels rose 200-300 pg/ml during CCK plus saline. This was slightly attenuated by glucose. During CCK plus amino acids, the PP response was augmented to 600-800 pg/ml. Since secretin is also released after the ingestion of a meal and intraduodenal acidification is a potent stimulus not only of secretin but also of gastric and pancreatic SLI release, the effect of secretin was examined additionally.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of low-dose somatostatin infusion on pancreatic and gastric endocrine function in lean and obese nondiabetic human subjects.

The present study was designed to compare, in lean and obese nondiabetic subjects, basal and postprandial levels of peripheral venous plasma insulin, glucagon, gastrin, pancreatic polypeptide (PP), glucose, triglycerides, and somatostatin-like immunoreactivity (SLI) during the infusion of synthetic somatostatin-14 or saline. Thirty-five minutes before the ingestion of the test meal, an infusion of synthetic somatostatin-14 was started at a rate of 0.5 ng/kg X min and was increased to 1.0 ng/kg X min 30 min after consumption of the meal and lasted for another 90 min. During the infusion of saline, basal peripheral vein levels of insulin, gastrin, and triglycerides were elevated in obese subjects, whereas basal plasma SLI levels were significantly lower compared with the lean controls. Basal glucagon and PP levels were similar in both groups. After the ingestion of the meal, augmented concentrations of insulin and gastrin were observed in the obese subjects, whereas postprandial SLI and PP levels were reduced. Chromatography of fasting plasma revealed all measurable SLI to be confined to the void volume fractions of a Bio-Gel P-10 column. The rise in SLI after the meal was due to an increase of SLI co-eluting with somatostatin-28 and somatostatin-14. During the infusion of somatostatin, only basal insulin levels were significantly lower in the obese subjects, whereas no change of any basal hormone level was observed in the lean group. During the infusion of somatostatin, SLI levels were elevated by 20-30 pg/ml in both groups compared with the saline controls. During the infusion rate of 0.5 ng/kg X min, only postprandial PP levels were reduced significantly in the obese group, while all the other parameters were unaffected in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

v- and t-SNARE protein expression in models of insulin resistance: normalization of glycemia by rosiglitazone treatment corrects overexpression of cellubrevin, vesicle-associated membrane protein-2, and syntaxin 4 in skeletal muscle of Zucker diabetic fatty rats.

Insulin stimulation of adipose and muscle cells results in the translocation of GLUT4 from an intracellular location to the plasma membrane; this translocation is defective in insulin resistance. Studies have suggested an important role for synaptobrevin and syntaxin homologues in this event, particularly the v-soluble N-ethylmaleimide attachment protein receptors (SNAREs) cellubrevin and vesicle-associated membrane protein-2 (VAMP-2) and the t-SNARE syntaxin 4, but the expression of these proteins has not been studied in insulin-resistant tissues. Therefore, we examined SNARE protein content in skeletal muscle from Zucker diabetic fatty (ZDF) rats compared with lean controls and determined the effect of the thiazolidinedione insulin sensitizer rosiglitazone on these proteins. GLUT4 levels in skeletal muscle from ZDF rats were similar to those in lean control animals. In contrast, cellubrevin, VAMP-2, and syntaxin 4 protein levels were elevated (2.8-fold, P = 0.02; 3.7-fold, P = 0.01; and 2.2-fold, P < 0.05, respectively) in skeletal muscle from ZDF rats compared with lean controls. Restoration of normoglycemia and normoinsulinemia in ZDF rats with rosiglitazone (30 micromol/kg) normalized cellubrevin, VAMP-2, and syntaxin 4 protein to levels approaching those observed in lean control animals. These data show that elevated v- and t-SNARE protein levels are associated with insulin resistance in skeletal muscle and that these increases may be reversed by rosiglitazone treatment concomitant with a restoration of glycemic control. Such increases in SNARE protein levels were not observed in streptozotocin-induced diabetic rats, which suggests that hyperinsulinemia rather than hyperglycemia may be more important in modulating SNARE protein expression in rodent models of insulin resistance. Consistent with this hypothesis, elevated levels of SNARE proteins were also observed in 3T3-L1 adipocytes chronically treated with insulin (500 nmol/l for 24 h). These data argue that SNARE protein levels may be altered in insulin-resistant states and that the levels of these proteins are modulated by agents that increase insulin sensitivity. Moreover, these data demonstrate for the first time altered expression of proteins known to regulate GLUT4 translocation in a model of diabetes.

Immunologic properties of biosynthetic human insulin in vitro.

Some immunologic properties of biosynthetic human insulin (BHI) were examined in vitro. An identical behavior was found for BHI and pancreatic human insulin as standard preparation and for A14-mono-labeled BHI and pork insulin as tracer in the insulin radioimmunoassay. BHI proved to be free of human proinsulin and C-peptide. Insulin antibodies in serum of two diabetic patients showed a preferential binding of 125I-bovine insulin. However, the antibody titers were almost identical for A14-mono-labeled BHI and pork insulin. These studies did not reveal any characteristic immunologic properties of BHI compared with highly purified pancreatic human and pork insulin.

Circulating amino acids and pancreatic endocrine function after ingestion of a protein-rich meal in obese subjects.

We measured plasma amino acid together with insulin, glucagon, pancreatic polypeptide (PP), and glucose concentrations after the ingestion of a protein meal in lean and obese subjects. The basal plasma amino acid levels were similar in both groups. The postprandial increase in the plasma amino acid levels in the obese subjects was only 15-50% of that in the lean subjects. The mean basal and peak postprandial plasma insulin levels were significantly higher (72 and 165 pmol/L) in the obese group than in the lean group (36 and 115 pmol/L; P less than 0.05-0.01). The postprandial rise in plasma glucagon was largely attenuated in the obese subjects, and there was no difference in plasma PP and glucose levels in the 2 groups. To further evaluate the role of circulating amino acids on pancreatic endocrine function in obese and lean subjects, an amino acid mixture consisting of 15 amino acids was infused iv. During the infusion the plasma amino acid levels were comparable in both groups. Plasma insulin rose by 36 +/- 7 (+/- SE) pmol/L (5 +/- 1 microU/mL) in the lean and 129 +/- 22 pmol/L (18 +/- 3 microU/mL) in the obese subjects, whereas plasma glucagon, PP, and glucose levels were similar in both groups. In view of the 3.6-fold greater insulin responses in the obese subjects, it is likely that circulating amino acids contribute to their hyperinsulinemia in spite of the reduced postprandial rise of amino acids in this group (50-85%). Thus, under physiological conditions amino acids have to be considered as an important regulatory component of postprandial insulin release in obese subjects.

Periodic hypersomnia: case report with biochemical and EEG findings.

We report on a 23-year-old patient with periodic hypersomnia. Electroencephalographic (EEG) background activity for this individual was slightly slowed in the EEG during an episode of hypersomnia, and intermittent slow activity was found in addition. Usual laboratory parameters were normal; however, leucine-enkephalin was markedly elevated in the plasma at that time, whereas free cysteine could not be demonstrated. Clinical findings were normal in the following years, and the EEG background activity returned to normal; leucine-enkephalin and cysteine also returned to normal values.

What is the role of the supplementary motor area in movement initiation?

The hierarchical position of the supplementary motor area (SMA) relative to the primary motor cortex is discussed on the basis of neurological observations and of animal experiments. In the last 10 years evidence has accumulated, especially from studies on the human brain, that the supplementary motor area is a hierarchically superior structure involved in the processes of movement initiation. Single unit studies in subhuman primates also revealed neuronal populations related to aspects of movement preparation rather than to the movement per se. However, we report that a surprisingly large subpopulation of SMA neurones has features classically found in the primary motor cortex (MI). These MI-like neurones precede movement onset by a relatively short interval. The occurrence of such "short-lead neurones" was somewhat higher in MI, but the histograms of lead-times were completely overlapping in the two areas. Taken together with the fact that the SMA is microexcitable and is part of the origin of the pyramidal tract, these findings suggest that the SMA functions also in parallel with MI as concluded by Woolsey and coworkers (1952). Finally, the SMA and MI are reciprocally interconnected, a situation which is not unlike that of the cortical visual areas.

Effect of naloxone on pancreatic and gastric endocrine function in response to carbohydrate and fat-rich test meals.

The present study was designed to determine the effect of naloxone, a specific opiate receptor antagonist, on postprandial levels of insulin, glucagon, pancreatic polypeptide (PP), somatostatin-like immunoreactivity (SLI) and gastrin in response to carbohydrate and fat-rich test meals in a group of 6 healthy volunteers. The addition of naloxone to a meal consisting of 50 g sucrose dissolved in 200 ml water augmented the rise of plasma insulin levels significantly during the first 30 min after its ingestion and reduced the rise in plasma insulin and pancreatic polypeptide and elevated glucagon levels during the last 30 min of the experimental period. When sucrose was dissolved in 200 ml cream the addition of naloxone augmented the postprandial rise of insulin levels between 15 and 60 min after ingestion of the meal and elicited an increase of plasma SLI and PP levels throughout the entire experimental period which indicates that post-prandial levels of insulin, glucagon, PP and SLI are modulated via endogenous opiate receptors during the ingestion of carbohydrate and fat test meals and that this effect depends on the composition of the ingested nutrients. These data raise the possibility that endogenous opiates participate in the regulation of postprandial insulin, glucagon, somatostatin and pancreatic polypeptide release not only in certain disease states as demonstrated recently for insulin secretion in type II diabetes mellitus but endogenous opiates may also be of importance under physiological conditions.

Effect of physiological increments of blood glucose on plasma somatostatin and pancreatic polypeptide levels in dogs.

The present study was designed to determine the effects of physiological increments of plasma glucose levels upon basal and stimulated plasma somatostatin and pancreatic polypeptide levels. In seven conscious dogs the elevation of plasma glucose levels by 30-40 mg/dl did not change basal somatostatin and pancreatic polypeptide levels. During stimulation of these two hormones by acetylcholine and the octapeptide of cholecystokinin intravenous infusion of glucose elicited a significant decrease of somatostatin levels by 30 pg/ml and of pancreatic polypeptide levels by 300 pg/ml. The present data demonstrate that a physiological elevation of plasma glucose levels inhibits stimulated but not basal somatostatin and pancreatic polypeptide levels which may be of importance for nutrient entry and metabolism.

Carbohydrates modulate opiate receptor mediated mechanisms during postprandial endocrine function.

The present study was designed to determine the role of carbohydrates during naloxone-induced opiate receptor blockade upon the postprandial rise of plasma somatostatin (SLI), insulin and pancreatic polypeptide (PP) levels in response to protein and fat test meals in conscious dogs. Test meals consisting of 50 g liver extract + 50 g sucrose or 50 g corn oil + 50 g sucrose dissolved in 300 ml water were instilled intragastrically, respectively. Additionally, liver extract and fat meals were given with a concomitant intravenous infusion of glucose. To all test meals either naloxone (4 mg) or saline was added. The addition of sucrose to liver extract or the infusion of i.v. glucose during the liver meal abolished the inhibitory effect of naloxone on the rise of postprandial somatostatin levels which has been described recently. The addition of carbohydrate either orally or intravenously to the fat meal resulted in an even stimulatory effect of naloxone upon the rise of postprandial somatostatin levels. Insulin levels were not changed during liver extract + sucrose or i.v. glucose, respectively. When sucrose or i.v. glucose was administered together with the fat meal the addition of naloxone augmented postprandial insulin secretion. Pancreatic polypeptide (PP) release was augmented during the combination of sucrose or i.v. glucose with the fat and liver meal when naloxone was present in the meals. The present data demonstrate that the addition of carbohydrates either orally or intravenously to fat and protein meals modulates the effect of endogenous opiates in the regulation of postprandial somatostatin, insulin and pancreatic polypeptide release in dogs in a way that carbohydrates induce inhibitory mechanisms that are mediated via endogenous opiate receptors.

Evidence for a role of endogenous opiates in postprandial somatostatin release.

Previously, we have demonstrated the effects of exogenously administered opiates on somatostatin release in dogs and therefore the present study was designed to determine the effect of endogenous opiates via naloxone-induced opiate receptor blockade on somatostatin release. Additionally, plasma insulin and pancreatic polypeptide (PP) levels were determined in response to intragastrically instilled protein, carbohydrate and fat test meals in a group of eight conscious dogs. To all test meals either naloxone (4 mg) or saline was added. The rise of plasma somatostatin levels in response to liver extract, sucrose and fat was attenuated significantly by naloxone. Naloxone had no effect on the rise of postprandial plasma insulin and PP levels. The present data demonstrate that endogenous opiates have a stimulatory effect on postprandial somatostatin release in dogs which indicates a tight interaction that might be of relevance for nutrient homeostasis.

A fast protein liquid chromatography (FPLC) method for study of thyrotropin-releasing hormone (TRH) and its metabolite histidyl-proline diketopiperazine (CHP) in human blood: degradation in liver and pancreatic diseases.

We have developed a convenient method combining fast protein liquid chromatography (FPLC) with sensitive radioimmunoassay (RIA) for thyrotropin-releasing hormone (TRH) to separate and identify TRH and its metabolite histidyl-proline diketopiperazine (CHP) and applied this to study inactivation of TRH by blood extracts from patients with liver cirrhosis (LC) and acute edematous pancreatitis (AP). Blood samples spiked with TRH and CHP were extracted by cold methanol and injected on a reverse-phase FPLC column. A linear gradient was applied for separation. Subsequent analyses of fractions by RIA for TRH revealed that only fractions 9-10 contained TRH. Separation by retention time (9.9 +/- 0.8 min for TRH, 10.5 +/- 0.6 min for CHP, mean +/- SEM) was highly reproducible. For degradation studies, pooled sera from patients with LC and AP were incubated with TRH and CHP for 60 min. Inactivation of TRH was less rapid in the presence of blood extract from LC patients than that from normal subjects or AP patients. CHP was more stable than TRH. These data suggest that activity of TRH-degrading enzymes is reduced in liver disease, whereas it does not appear to be altered in AP. Degradation of CHP does not closely reflect metabolic processing of its major precursor. This rapid and sensitive method may be applicable for further investigations on the metabolism of TRH in organic fluids.

Acoustic imprinting in guinea fowl chicks: age dependence of 2-deoxyglucose uptake in relevant forebrain areas.

In 7-day-old guinea chicks play back of an imprinted acoustic stimulus was previously found to correlate with increased uptake of 2-deoxyglucose (2DG) in 3 rostral forebrain areas (HAD, LNH and MNH). Subdivisions of these areas defined by 2DG labelling may be association fields. Auditory areas did not show labelling differences between imprinted and control animals. In the present study imprinted guinea chicks of different age and with different experience were used in the 2DG experiments. Seven-day-old chicks (beyond the sensitive phase), 4-day-old (at the decline of sensitive phase) and 1- and 3-day-old chicks (within the sensitive phase) were given a 2DG injection and afterwards heard the stimulus to which they had been imprinted previously (1.8 kHz, 3 tone pips per s). While the typical labelling pattern was weak or absent in 1-4-day-old chicks the older animals consistently had high 2DG uptake in these areas. Unsuccessfully imprinted 7-day-old chicks, having all the behavioral test experience, showed no or weak labelling. These results are related to current literature on reticular activation of the relevant areas and on morphological changes there with termination of the sensitive phase. It is argued that the reticular activation of these areas, e.g. attention mechanisms are instrumental in securing imprinting success and that subsequent reorganization of the areas leads to stronger metabolic activation after the sensitive phase.

Effect of CCK on insulin, glucagon, and pancreatic polypeptide levels in humans.

The present study was designed to determine the effect of low doses of cholecystokinin (CCK) on insulin, glucagon, and pancreatic polypeptide (PP) secretion in the basal state and during prestimulation with amino acids and glucose alone or in combination. Two different amino acid solutions available for use in humans were employed. Aminosteril-N-Hepa was better for the imitation of the so-called "insulinogenic" amino acids while Aminoplasmal L-10 gave more comparable plasma levels of the "glucagonogenic" amino acids as observed after a protein-rich meal. In healthy volunteers, low-dose CCK infusion [Thr28,Nle31-CCK 25-33 (CCK-9)] in stepwise increasing doses of 5, 10, and 20 pmol/kg/h had no effect on basal, glucose-, or amino acid-stimulated insulin release. During the combination of Aminoplasmal + glucose, there was a small and only transient increase of plasma insulin levels that did not occur during Aminosteril + glucose. CCK did not alter glucagon levels either during i.v. amino acids alone or during combination of amino acids with glucose. CCK-stimulated PP levels in the basal state in a dose-dependent manner. This effect was enhanced during i.v. Aminosteril but not i.v. Aminoplasmal infusion. During i.v. glucose, the effect of CCK on PP levels was abolished. In conclusion, the present data demonstrate that CCK is unlikely to be a stimulus of insulin and glucagon secretion in the basal state and also during prestimulation by fairly physiological quantities of amino acid mixtures. On the other hand, the present data support a physiological role of CCK in the regulation of PP secretion.

Role of amino acids in stimulation of postprandial insulin, glucagon, and pancreatic polypeptide in humans.

Protein-rich meals stimulate secretion of insulin, glucagon, and pancreatic polypeptide (PP) from the endocrine pancreas. On the one hand, this is due to increased levels of circulating amino acids, and, on the other, neural and/or endocrine factors can contribute to activation of islet cell function. The present study was designed to determine, first, pancreatic endocrine function and postprandial amino acid levels after a protein and a protein-carbohydrate meal and second, insulin, glucagon, and PP levels during infusion of amino acid mixtures that imitate the postprandial amino acid pattern. In healthy volunteers the ingestion of a protein-rich meal (300 g tenderloin steak) elicited within 1 h an increase of virtually all amino acids by 20-400 mumol/L above basal values. The infusion of two different amino acid solutions available for use in humans showed that Aminosteril-N-Hepa (AS) was better for the imitation of the so-called "insulinogenic" amino acids while Aminoplasmal L-10 (AP) gave more comparable plasma levels of the "glucagonogenic" amino acids. Both solutions were not able to imitate the postprandial amino acid pattern completely. With regard to insulin levels, both solutions gave a comparable increase, while AP but not AS stimulated glucagon and PP levels. This suggests that circulating amino acids may be responsible for 60% of the postprandial insulin response after a protein meal, while their contribution to glucagon release can only be roughly estimated at 30-60%. The contribution of circulating nutrients to the greater insulin response after the protein-carbohydrate meal was comparable (60%), while the attenuated glucagon response can be ascribed almost completely to the effect of circulating nutrients. In conclusion, the present data demonstrate that the composition of amino acid mixtures is as yet not ideal for a complete imitation of the postprandial amino acid pattern. The insulin, glucagon, and PP response depends on the amino acid mixtures and accordingly the respective plasma amino acid concentrations obtained during infusion studies. The adequate imitation of plasma amino acid levels is of critical importance for the evaluation of absorbed and circulating amino acid effects in the postprandial state.

Contribution of postprandial amino acid levels to stimulation of insulin, glucagon, and pancreatic polypeptide in humans.

The present study was designed to examine the contribution of the postprandial increase of plasma amino acids after ingestion of a protein-rich meal to the rise of the three pancreatic hormones insulin, glucagon, and pancreatic polypeptide (PP). A mixed amino acid solution was designed, which permitted a fairly close imitation of the arterial plasma pattern of the 21 amino acids that rise after ingestion of a 200-g porcine steak meal. In 10 healthy subjects the intravenous infusion of this mixed amino acid solution at a rate of 10 g/h elicited a rise of the 21 amino acids examined that correlated well with the postprandial increase (r = 0.89, p < 0.001). The maximal rise of plasma insulin (64 +/- 5 pmol/L) and glucagon (630 +/- 21 ng/L) was not significantly different from the postprandial increase of these two hormones (49 +/- 4 pmol/L and 780 +/- 28 ng/L, respectively). PP levels rose by 316 +/- 33 ng/L postprandially, which was clearly above the increase of 112 +/- 13 ng/L during intravenous amino acids (p < 0.01). In conclusion, the present data demonstrate that the postprandial rise of amino acid levels in arterialized venous plasma can account for most if not all of the postprandial increase of insulin and glucagon during the ingestion of a protein-rich meal. In contrast, only 35% of postprandial PP levels can be ascribed to the rise of plasma amino acids. In contrast to the effect of carbohydrate-rich meals, an enteric augmentation of insulin release seems to be of minor and possibly of no importance during ingestion of protein-rich meals.