Brain histidine and food intake in rats fed diets deficient in single amino acids.

Histidine (His) is elevated in plasma and brain during protein deficiency as well as in several pathological conditions, leading to the possibility of a direct effect on central nervous system (CNS) function. In this study, groups of weanling rats were fed diets containing graded levels of casein or a single indispensable amino acid (IAA: Leu, Val, Ile, Phe, Trp, Thr, Met or Lys) in order to produce nutritionally-deficient states. Body weight gains and food intakes were recorded daily for 2 wk. Whole brain and serum samples were obtained and analyzed for amino acid (AA) content. All weight gain and food intake responses could be predicted by the Saturation Kinetics Model. The only consistent pattern observed in AA profiles which could be correlated with food intake was an increase in brain His concentrations. Limiting dietary casein or IAA elevated brain His above controls 2.5- and 1.5-fold, respectively. Food intake was generally depressed by 50% at brain His concentrations above 105 nmol/g. Since His is the precursor of the depressant neurotransmitter histamine (HA), systemic increases may be significant in that HA could be a possible cause of the anorexia observed in protein and IAA deficiency.

Use of the peroxidase-antiperoxidase technique for differential staining of multiple cell types in the rat pancreatic islet.

Multiple staining of the endocrine cells of the pancreatic islet was studied in tissue obtained from adult rats. After fixation in Bouin's fluid and processing for light microscopy, the unlabeled peroxidase-antiperoxidase (PAP) technique was performed. Successful staining procedures used variations of the PAP technique with 3,3'-diaminobenzidine (DAB) and 4-chloro-1-naphthol (CN) as chromagens. The purpose of this study was to stain as many of the four primary cell types (A-cells, B-cells, D-cells, PP-cells) as possible either simultaneously or sequentially using photomicroscopy. At optimum antibody titer, there was minimal nonspecific background staining which made it possible to differentiate cell types by intensity of the chromagen. Any two cell types can be shown by using DAB with the first antibody and CN with the second. To demonstrate three cell types simultaneously, three methods which altered dilutions and chromagens were used. The first method consisted of decreasing dilutions of primary antibody with DAB and CN as the chromagens. The second method involved repetitive DAB applications resulting in three intensities of brown. The third method used a DAB immersion after the second cell type was stained. This produced a color differential so the third cell type could be distinguished with CN. To demonstrate the three cell types sequentially, a masking technique was introduced with photomicroscopy. In order to block the preceding complex, the previous cell type (demonstrated by CN) was restained with DAB at an increased dilution. The next cell type was then stained with CN. These four methods were tried in attempts to stain four cell types in the same tissue section.

Control of physiological response in the rat by dietary nutrient concentration.

In three separate experiments, growing, male Sprague-Dawley rats were fed diets which contained: 1) graded levels of fiber 0-70%, 2) graded levels of pyridoxine 1-10 mg/kg diet, and 3) graded levels of casein 0-30%. The following physiological responses were measured in each respective experiment: 1) food intake, weight gain, serum triglycerides, 2) food intake, weight gain, SGPT levels, and 3) weight specific food intake, weight gain, relative testes weight. Diets were fed as a single source, and in each case, physiological response could be predicted as a function of dietary nutrient concentration. When self-selection is prevented, rats establish new steady-state response profiles, which are sigmoidal in shape and dependent on the interaction of the rats' genetic potential with the environmental configuration.

Kinetics of specific food intake and weight gain in rat.

We report the expanded application of a recently developed time-invariant analytic function that relates food intake, growth (weight gain), and dietary nutrient concentration in the laboratory rat. Data from seven experiments were utilized, providing analysis of three stages of rat (Sprague-Dawley) growth (weanling, adult, and pregnant) and a variety of nutrients: casein, lactalbumin, amino acid mix, thiamin, and pyridoxine. For each experiment the rats were fed graded levels of a nutrient, ranging from 0% to a percentage well above the recommended amount, in an otherwise nutritionally adequate diet. Weight-specific weight gains and food intakes were calculated for each dietary group and fitted to the Gompertz equation. The resulting rate constants for each group were identical for weight gain and food intake. The rate constants were then fitted as functions of dietary nutrient concentration by the four-parameter mathematical model for physiological responses. Kinetic rate constants could be characterized as functions of the dietary concentration of each nutrient tested. This approach offers new possibilities for the determination and optimization of dietary requirements. With this approach, one may characterize food intake and growth in a single rate constant, which varies as a function of dietary nutrient concentration.

Regional distribution of pancreatic polypeptide cells in the 21-day fetal rat pancreas.

21-day fetal rat pancreata were stained with the unlabeled antibody peroxidase-antiperoxidase technique using bovine pancreatic polypeptide as the primary antibody. Total counts of pancreatic polypeptide cells were made over the entire pancreas. It was found that the head region contained the greatest number of pancreatic polypeptide cells with the body next and the tail having the smallest number. The pancreatic polypeptide cells of the body were concentrated in the portion closest to the distal duodenum. This distribution pattern seems to support the suggested role of pancreatic polypeptide on the physiological function of the digestive tract.

The ability to predict weight gain, individual organ weight, and corresponding food intake in the rat by the four-parameter model for physiological responses.

The applicability of the four-parameter model for physiological responses to the prediction of food intake and corresponding weight gain and individual organ weight gain was studied further in 40-day postpartum male rats. Seven groups of animals were maintained on diets in which protein content ranged from 0 to 23.54% casein. Food intake and weight gain were recorded every other day for each animal for 21 days. At the termination of the experiment the following organs were removed and weighed: liver, heart, lungs, spleen, kidneys, adrenals, and testes. When these weight values are fitted by use of the four-parameter model, food intake and total animal and organ weight gains can be predicted in relation to the amount of protein in the diet. It was found that liver, heart, lungs, spleen, and whole animal had similar K(0.5) values. However, it was also shown that there is variation in response of organs when relating organ weight as a percentage of body weight. For example, heart, lungs, and testes show an increased ratio on low protein diet while liver, kidneys, and adrenals maintain a fairly constant ratio and the spleen shows a decreased ratio. Additionally, it was noted that the animals on low protein diet consumed more food per gram body weight but did so at a slower rate. Possible future applications of the four-parameter model for physiological reponses are discussed.

Further evidence for an argyrophilic fourth cell type in the pancreatic islet of the rat.

An argyrophilic fourth cell type in fetal and adult rat pancreatic islets can be identifed by using a modification of the Grimelius silver statin. This cell is much more abundant in the fetal pancreas than in the adult. By employing the modified silver technique followed by restaining with the indirect immunofluorescent procedure for somatostatin, the content of this argyrophilic fourth cell was studied further. Comparison of these histochemical studies demonstrated that somatostatin was not located in the fourth cell of either the adult or fetal rat pancreas. These results indicate that the D-cell and the fourth cell type are not the same cell. Thus far the only product associated with this argyrophilic cell is pancreatic polypeptide. As a result this cell probably represents the PP-cell of the Wiesbaden classification.

Observations of esophageal relationships in a case of achalasia.

This study of an 85-year-old man with severe achalasia correlates postmortem findings with the medical history and clinical manifestations. Possible explanations are discussed and related to cases previously described.

Modification of an in vitro technique in order to grow adult rat anterior pituitary in organ culture.

The study of adult rat anterior pituitary maintained in vitro was made possible by a modification of the suspension system used in the watch-glass method of organ culture. The integrity and health of the tissue was observed with the electron microscope. Somatotrophs and gonadotrophs were seen routinely. A cell with large (400-750 nm) granules and thought to be a luteotroph (mammotroph) was also seen. These results support the use of this method for the growth of adult rat anterior pituitary, or other large explants, for short periods of time in organ culture.

The staining of pancreatic delta cells for light microscopy.

Three staining techniques--Hellerström-Hellman alcoholic silver nitrate, pseudoisocyanin, and the immunofluorescent technique for somatostatin--were compared to determine whether they are staining the same cell in the pancreatic islet of the rat. These comparisons were made by using staining and restaining procedures on the same islet. The results demonstrate that all three procedures stain the delta cell of the rat pancreatic islet. Some advantages of using pseudoisocyanin are discussed.

Localization of human pancreatic polypeptide in an argyrophilic fourth cell type in islets of the rat pancreas.

An argyrophilic fourth cell type in the rat pancreatic islet can be differentiated from other silver-staining cells by using a modification of the Grimelius aqueous silver nitrate technique. Restaining of the tissues using fluorescent techniques with anti-HPP (Human Pancreatic Polypeptide) serum results in bright fluorescence in the fourth cell type.

Innervation of the cultured fetal rat pancreas.

The fetal rat pancreas, explanted at 18 days of gestation and cultured up to ten days, contains numerous acetylcholinesterase-positive neurons. These nerves usually appear in small ganglia although single nerve cells are encountered. The axons of these intrapancreatic nerves appear to terminate only in the islet tissue and not on any exocrine components of the explant. It is concluded that the fetal rat pancreas contains an islet-specific group of cholinergic neurons.

A, B, D cells and a fourth cell type in long-term cultures of fetal rat pancreas.

Whole pancreases from fetal rats of 13 days and 18 days gestation were explanted onto rayon grids and grown in organ culture. Cultures were fixed in Bouin's fluid, sectioned and stained with the fluorescent antibody techniques for glucagon and insulin, aldehyde fuchsin for B cells, pseudoisocyanin for D cells and a silver technique for the fourth cell type. The 13-day explants were fixed after 10 days in culture. A, B and D and the fourth cell type were seen, indicating that precursors of all four endocrine cell types must be present in the fetal pancreas shortly after the formation of the pancreatic bud (11 days). Further, the presence of these four cell types in the walls of tubules in these cultures indicates the tubules as the site of origin of all the endocrine tissue. The 18-day explants were collected every other day of culture from 2 to 30 days in a long-term experiment. A number of large islets with well granulated B cells was still present after 30 days of culture. The relative abundance of cell types at different stages was estimated as follows: 18-day fetal controls, A greater than B=4 greater than D; after 2 to 10 days in culture, B greater than A greater than or equal to D; after 18 to 30 days in culture, B greater than D greater than A greater than 4.

Ontogeny of four cell types in fetal rat islets using histochemical techniques.

The pancreas of the fetal rat was collected from the first appearance of the pancreatic bud at 11 days of gestation, and every day thereafter until birth. After birth the neonatal pancreas was collected every day for one week, and at intervals thereafter. Fetal B-cells were stained with Gomori's aldehyde fuchsin at 16 1/2 days, and with the immunofluorescent technique for insulin at 14 days. The A-cells were stained as early as 13 days using the fluorescent antibody technique for glucagon. The D-cells first stained at 17 days with pseudoisocyanin. A 4th cell type was found which stained black with silver nitrate, using a method derived from the Grimelius technique for A-cells. This 4th cell type appeared at 15 days in the fetus, reaching its greatest abundance around 19 days, and then declined in numbers after birth until adulthood, when occasionally one or two cells were found.

Stains for A, B, and D cells in fetal rat islets.

Ten techniques often used for identification of A, B, and D cells in adult islets of Langerhans were applied to fetal rat pancreas. Modifications were tried with many of these techniques. Two indole methods (xanthydrol and postocoupled benxylidene reactions) and a cryostat technique using o-phthaladehyde failed to stain fetal islets. Phosphotungstic acid hematoxylin and lead hematoxylin lightly stained fetal A cell granules in Helly's fixed tissue. The Grimelius silver nitrate technique stains adult rat A cells but failed to stain fetal cells. A modification of this technique stained fetal A cells and a possible 4th cell type. The specificity of this method was confirmed by restaining stained cells with a fluorescent antibody technique and with pseudoisocyanin. B cells, as previously reported, were readily stained by the aldehyde fuchsin technique. Fetal D cells were not stained by the Hellerstrom-Hellman alcoholic silver nitrate method, nor did they display pseudoisocyanin metachromasia after acid hydrolysis; they did fluoresce brightly with this technique when viewed with UV light. It was thus possible to distinguish the three usual cell types, plus a possible fourth type, in the fetal rat pancreas.