Immunochemical studies of an insulin-like material in the parotid gland of diabetic BB rats.

Immunocytochemical and radioimmunoassay studies were performed on pancreatic and parotid tissues from diabetic BB and control Wistar rats. Compared with those of normoglycemic controls, the pancreata of diabetic BB rats generally lacked insulin-containing B-cells. Extracts from the parotid glands of diabetic rats contained less immunoassayable insulin-like material than was present in parotid extracts of controls. However, the parotid glands of both groups of animals contained numerous cells displaying insulin-like immunoreactivity. These insulin-immunoreactive cells, located mainly in the intercalated portion of the duct system, were comparable to those we reported recently in the parotid glands of normal and streptozocin-diabetic Sprague-Dawley rats. The presence of an insulin-like material in the parotid salivary gland of two types of diabetic animals suggests that such cells may be spared, in part, from the effects of both chemical and hereditary diabetogenic factors.

Immunocytochemical localization of insulin- and somatostatin-like material in human breast tumors.

Four types of human breast lesions and C3H mouse mammary adenocarcinomas (type A) were examined for the immunocytochemical localization of cells containing hormone-like substances. Insulin- or somatostatin-like immunoreactive material was observed in scattered single cells and nests of tumor cells in seven of eight infiltrating duct carcinomas, and in the majority of tumor cells from an anaplastic carcinoma. A few somatostatin-immunoreactive cells were observed in only one of seven fibroadenomas studied. No immunoreactive cells were observed in mouse adenocarcinomas or in human breast dysplasias. These results suggest that cells with hormone-like immunoreactivity may be a common feature in two types of malignant human breast tumors.

Immunoreactive hormones in human breast tissues.

Samples of breast tissue obtained at biopsy or mastectomy from women with benign breast disease and infiltrating duct or anaplastic carcinoma were maintained for 2 weeks in organ culture synthetic medium 199 without additional serum or hormones. Media were changed every 48 hours. Media withdrawn from the tissues were assayed for insulin, prolactin (Prl), and parathyroid hormone (PTH). In addition, tissue explants were extracted in acid-alcohol and assayed for insulin by standard radioimmunoassay (RIA) procedures. At day 0 portions of breast tissue from patients with malignant or benign disease were fixed in Bouin solution; they were then embedded in paraffin; and serial sections were obtained for histologic and immunocytochemical examination. The dissection media assayed for insulin and PTH by RIA showed that the hormones were present in media from patients with benign as well as malignant disease. However, there was no significant difference between the two groups of women. Only traces of Prl were detected in media. The amount of insulin present in certain tissue explants appeared to increase with time in culture. Immunocytochemical studies showed that insulin-like or PTH-like immunostaining appeared most often in malignant tumor tissue and was observed infrequently or not at all in patients with benign disease. Prl-positive cells were rare. These data suggest that breast tissues contain and may synthesize significant amounts of certain hormones that may influence the growth and proliferation of breast cells.

Carnitine content of skeletal and cardiac muscle from genetically diabetic (db/db) and control mice.

Portions of diaphragm and heart from genetically diabetic and control mice of three age groups were analyzed for free fatty acid, triglyceride, and carnitine content. Triglyceride levels were increased consistently in both cardiac and skeletal muscle from diabetic animals while the amount of free fatty acids was comparable to that measured in tissue from lean littermates. Free carnitine and total carnitine were decreased in diaphragm and heart from db/db mice throughout the course of the study. While the levels of short-chain carnitine were comparable in tissue from control and diabetic animals, the amount of the long-chain derivative was elevated significantly in both diaphragm and heart in the 18-week-old diabetic mice. The results are discussed with respect to (a) alterations in hepatic carnitine metabolism in this animal model reported previously by this laboratory, and (b) changes in carnitine metabolism which we observed in skeletal muscle from streptozotocin-diabetic animals.

A longitudinal and comparative study of some structural and hormonal alterations in the endocrine pancreas of spontaneously diabetic and streptozotocin-induced diabetic mice.

A comparative study of the endocrine pancreas was carried out in genetically diabetic (db) mice and in mice with streptozotocin-induced (Sz) diabetes over a 12-week period of pronounced diabetes. Mice were examined at 9, 12 and 21 weeks of age. Plasma and pancreatic levels of immunoreactive insulin and immunoreactive glucagon were measured in both experimental animal models, and the biochemical data obtained were correlated with ultrastructural observations on the endocrine pancreas. Both pancreatic and plasma immunoreactive insulin levels were severely depressed in all Sz mice. Although pancreatic immunoreactive insulin concentrations in db mice were consistently lower than control values, these animals displayed a hyperinsulinemia which gradually dropped to control levels by 21 weeks. Pancreatic immunoreactive glucagon levels in 12- and 21-week-old db mice were markedly lower than those found in either control or in Sz mice. However, both db and Sz mice in all age groups exhibited a marked and persistent hyperglucagonemia. Pancreatic islet tissue was examined concurrently in control and experimental animals. The ultrastructural changes occurring in the endocrine cells are reported and discussed with regard to the pancreatic and plasma levels of the hormones presently monitored and in light of other recent studies on these animal models.

Glycogen synthase and phosphorylase activities in skeletal muscle from genetically diabetic (db/db) mice.

Glycogen synthase and glycogen phosphorylase activities were determined in skeletal muscle from lean and genetically diabetic (db/db) mice. The activities of both forms of glycogen synthase and of glycogen phosphorylase in diabetic mice were found to be comparable to those activities observed in muscle tissue from lean animals. Enzyme activities in muscle from diabetic animals remained essentially unaltered throughout the 12-week period studied while glycogen content was consistently depressed, despite the presence of hyperglycemia, hyperglucagonemia and transient hyperinsulinemia. These findings suggest that an impairment in muscle glycogen synthase activation may exist in db/db mice in vivo and that skeletal muscle glycogen synthesis may be resistant to the action of insulin.

Studies on the utilization and mobilization of lipid in skeletal muscles from streptozotocin-diabetic and control rats.

Several aspects of lipid metabolism in the soleus and diaphragm muscles of streptozotocin-diabetic and control rats were investigated. The triglyceride content of both muscles was elevated in the diabetic state and the presence of increased intracellular lipid was confirmed by electron microscopy. In vitro glucose and palmitate oxidation studies showed that both types of muscle from the diabetic animals metabolized more fat than did the soleus and diaphragm from control rats. While isoproterenol alone produced a significant lipolytic response in both the soleus and diaphragm from control and diabetic animals, there was no difference in the percent increase in fatty acids released from muscles of diabetic rats compared to controls. However, the absolute difference was greater when the diaphragms were compared. Muscles from experimental and control animals showed a marked reduction in the amount of free fatty acids released in response to insulin. In addition, in the presence of the hormone, both the absolute and percent isoproterenol-stimulated increases in fatty acids were significantly greater for both diaphragm and soleus muscles from diabetic rats. The effects of insulin, isoproterenol, and the combination of these two hormones on the amount of glycerol released into the incubation medium were similar to those found on free fatty acid release. The results of these experiments show that there is an apparent increase in fat utilization in skeletal muscle of diabetic rats. Furthermore, measurements of triglyceride concentration and the enhanced response to isoproterenol stimulation in the muscles from these animals suggests that they may have an increased capacity for mobilization of intracellular lipids. Finally, in the diabetic state, both the soleus and diaphragm appear to demonstrate an increased response to the antilipolytic effect of insulin as measured by the decreased amount of fatty acid released into the incubation medium, the percent change also being significant for the soleus muscle.-Stearns, S. B., H. M. Tepperman, and J. Tepperman. Studies on the utilization and mobilization of lipid in skeletal muscles from streptozotocin-diabetic and control rats.

Studies on serum lipids, insulin, and glucagon and on muscle triglyceride in rats adapted to high-fat and high-carbohydrate diets.

A comparisonwas made of lipid circulation, storage, and mobilization in rats adapted to lard or glucose diets. In the morning, lard-fed rats had higher blood triglyceride (TG) and free fatty acid (FFA) levels. In the evening TG was higher, but FFA was significantly lower in the lard vs. the glucose group. Fasting did not produce the characteristic increase in blood FFA in the lard-fed rats but was associated with a severe drop in their serum TG. Circulating glucose and insulin were not affected, while glucagon levels were increased by lard feeding. Nicotinic acid decreased fasting FFA levels to a greater extent in the glucose-fed rats. It was concluded that lard feeding depresses mobilization of fat from adipose tissue; on the other hand, it was found to increase storage and utilization of muscle TG. Fat feeding increased diaphragm TG concentrations threefold, as well as the number and size of intracellular fat droplets at the light and electron microscopic levels. Fasting decreased diaphragm TG in both groups, but the amount lost was greater in the fat-fed rats. Also, in vitro basal isoproterenol-stimulated (1 microgram/ml) FFA release by the incubated diaphragm was higher in the lard-fed group.

Glucagon and insulin relationships in genetically diabetic (db/db) and in streptozotocin-induced diabetic mice.

An inappropriate molar ratio of circulating insulin to glucagon is frequently associated with the metabolic alterations accompanying diabetes mellitus. Plasma immunoreactive insulin (IRI) and immunoreactive glucagon (IRG) levels were determined and the IRG:IRI ratio calculated at various intervals in overt diabetes in genetically diabetic (db/db) and in streptozotocin-treated mice. Plasma IRI levels in genetic mutants are elevated at nine weeks of age, but are comparable to values found in lean littermates by 21 weeks. The presence of a prevailing hyperglucagonemia is established for the first time in the intact db/db mice. Streptozotocin diabetics are found to have characteristically low plasma IRI and high plasma IRG values. The hormonal imbalance present in these two experimental animal models is accentuated when the data are expressed as the IRG:IRI ratio, which is seen to increase with the progression of diabetes.

Functional differentiation of the chick endocrine pancreas. II. The alpha cells and glucagon.

A radioimmunoassay for glucagon, together with electron microscopic observations of early embryonic alpha cells were utilized to examine developmental aspects of glucagon accumulation and release in the chick embryo. Immunoreactive glucagon was detected in both the pancreas and blood plasma from the fifth embryonic day onwards. In addition, emiocytotic events were observed in alpha cells as early as the fifth embryonic day. The early appearance of glucagon and its subsequent developmental profile correlate well with major events in carbohydrate metabolism occurring in the embryonic chick, and are discussed in relation to a functionally responding system, the developing liver. The present data show that glucagon is secreted at earlier embryonic stages than hitherto demonstrated, and suggest a developmental role for glucagon in hepatic glycogen metabolism.

Glycogen metabloism in the developing chick glycogen body: functional significance of the direct oxidative pathway.

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphatase were quantitatively determined for the first time in glycogen body tissue from late embryonic and neonatal chicks. For comparative purposes, the activities of these enzymes were examined also in liver and skeletal muscle from pre- and post-hatched chicks. The present data show that both the embryonic and neonatal glycogen body lack glucose-6-phosphatase, but contain relatively high levels of glucose-6-phosphate dehydrogenase. The activity of each dehydrogenase in either embryonic or neonatal glycogen body tissue is two- to five-fold greater than that found in muscle or liver from pre- or post-hatched chicks. The relatively high activities observed for both dehydrogenases in the glycogen body, together with the absence of glucose-6-phosphatase activity in that tissue, suggest that the direct oxidative pathway (pentose phosphate cycle) of glucose metabolism is a functionally significant route for glycogen utilization in the glycogen body. It is hypothesized that the glycogen body is metabolically linked to lipid synthesis and myelin formation in the central nervous system of the avian embryo.

Radioimmunological evidence for early functional activity in chick embryonic alpha cells.

A radioimmunoassay for glucagon was utilized to investigate early developmental aspects of glucagon synthesis and release in the chick embryo. Immunoreactive glucagon was detected in both the pancreas and blood plasma from the fifth embryonic day onwards. The present data show that the chick embryonic alpha cell has the potential for secretory activity very early in development, and suggest a developmental role for glucagon in hepatic glycogen metabolism.

Rh-D antibody titer in amniotic fluids. Its use as an index of the severity of erythroblastosis fetalis.

PIP: Report of a study of Rh antibody titers in the amniotic fluids from 42 Rh-negative sensitized pregnancies is presented. Amniocentesis was performed in the pregnant Rh-negative women having serum Rh antibody titers (indirect Coombs) ranging from 1/64 to 1/4,096. Spectrophotometric analysis was done on the antiRh-D titers. In order to predict the outcome of pregnancy based on the amniotic fluid Rh antibody content, all fetuses were divided into 3 categories. Those with titers of 1/4 or below should correspond to Category I; unaffected or mildly affected babies; those with titers of 1/8 to 1/16 should be in Category II or have moderate to severe disease; while fetuses with titers of 1/32 or above would be in Category III indicating very severe disease. Out of 18 cases with titers of 1/4 or below, 17 were unaffected or mildly affected. In 10 patients titers of 1/8 or 1/16 were observed as the highest titer. 8 infants in this group were moderately to severely affected. 14 cases were observed with titers of 1/32 or above and in 11 of them, the pregnancy outcome was a severely ill or dead infant. The other 3 cases in this group were babies with severe anemia who required 2 or 3 exchange transfusions. It was learned that the antibody titer of amniotic fluid of Rh-sensitized pregnancies can be used as a reliable index of the severity of hemolytic disease in the fetus.^ieng