Padmakar K. Dixit

Decreased breaking strength of diabetic rat bone and its improvement by insulin treatment

SummaryA simple instrument is described which measures the breaking strength of rat bones. The apparatus yields reproducible results and is suitable for use in measuring the strength of bones from both large and small animals.Diabetic rat femurs were more fragile and required less force to break in contrast to those from diabetic rats treated with insulin or normal rats. Daily insulin treatment significantly improved the bone cortical thickness and enhanced their capacity to withstand pressure, although these did not reach the level of the normal controls. The amount of force required to break the bone appears to be related to its cortical thickness and mass.

Insulin Content of Microdissected Fetal Islets Obtained from Diabetic and Normal Rats

1. “Insulin” content of microdissected islets was determined using the epididymal fat pad assay method. Measurements were made on fetal islets obtained from both normal and diabetic mothers and from normal postnatal rats up to five weeks of age. 2. The insulin content of the normal fetal islets, expressed as units “insulin” per gram dry weight of tissue, increases progressively with increasing fetal age; at birth the value approximates that of the adult. 3. The insulin content of the fetal islets obtained from diabetic mothers is between 7 and 19 per cent of the corresponding normal value. 4. In the acid-alcohol extracts of the pancreas of seventeen-day-old fetuses, small though measurable amounts of “insulin” were detected viz.: 0.23 units per gram wet weight of normal pancreas. There was a progressive increase in the extractable “insulin” content of the fetal pancreas with age; the “insulin” content of the twenty-one-day normal fetal pancreas was 5.3 units per gram of tissue. In the pancreas of the fetuses from diabetic mothers, the values were about one third of those in the normal. 5. Thus measurable amounts of insulin-like activity were found in the pancreatic tissue of seventeen-day-old fetuses; this is prior to the time that beta cell granules could be detected cytologically using the aldehyde fuchsin staining method. 6. The insulin content of the islet tissue remained relatively constant during the first five weeks of postnatal development.

Citrate metabolism in diabetes. I. Plasma citrate in alloxan-diabetic rats and in clinical diabetes.

Abstract A “bimodal” increase in plasma citrate levels was observed in alloxan-diabetic rats. Three days following alloxan injection, the plasma citrate levels in cardiac puncture blood was 9.40 mg. 100 ml. as compared to 4.95 for the control. At 14 days the value approximated that in the normal rats, whereas at 28 days (and subsequent time periods), the citrate levels were markedly elevated. In a second series of experiments in which the animals were killed by decapitation, a similar “bimodal” change in plasma citrate values was noted; however, the plasma citrate levels in both diabetic and control rats were generally higher than was the case when blood was drawn by cardiac puncture. No relationship could be established between the blood sugar level and plasma citrate values. In treated human diabetics, plasma citrate values were not significantly different from the normal, i.e., 2.70 as compared to 2.41 mg. 100 ml. However, in 2 cases of untreated human diabetes exhibiting severe ketoacidosis, the plasma citrate values were markedly elevated. In one, the initial value was 44.4 mg. 100 ml. serum; the level rapidly returned toward normal; at 4 weeks it was 1.56 mg. 100 ml. The increased serum citrate levels were not the result of citrate mobilization from bone, since there was no concomitant increase in the serum calcium levels. These results suggest the possibility that there may be an alteration in the tricarboxylic acid cycle in diabetes (both experimental and clinical), and that this alteration results in the accumulation of plasma citrate.

Decreased insulin secretion by isolated pancreatic islets from rats fed 4% protein diet.

Abstract Male weanling rats were fed either 4 or 16% (control) protein diets for 10 to 12 weeks. The animals were administered an i.v. injection of 30% glucose solution (3 g/kg body wt) and glucose tolerance was determined. The glucose tolerance in rats fed low protein diets was not significantly different from that of the control rats, indicating that clearance of high blood glucose levels in the animals fed low protein diets was not affected. Using a collagenase digestion technique, islets of Langerhans were isolated from another set of animals fed 4% or control diets. When these islets were perifused with a medium containing glucose, there was a typical biphasic insulin secretory response. The isolated islets from low protein fed rats exhibited considerably low insulin secretory response when exposed to medium containing 16.7 mM glucose (300 mg/dl), although no difference was found in the pancreatic insulin content of normal and low protein fed rats. This indicates that decreased insulin secretion could not be due to a decreased available pool of insulin. When glucagon (5 μg/ml medium) or arginine (20 mM) was added to a medium containing 8.4 mM glucose (150 mg/dl), the in vitro insulin release by the islets obtained from protein malnourished and control rats was enhanced. Again the control rat islets secreted much greater amounts of insulin. Insulin secreted by the islets in response to glucagon (5 μg/ml medium) was greater than that secreted in response to 16.7 mM glucose. It is suggested that the decreased insulin secretory response of islets from protein malnourished animals may be due to a diminished number of glucose recognition sites on the beta cells.

Citrate metabolism in diabetes: II. Tissue citrate content, citrate synthase and oxidation in alloxan-diabetic rat☆

Abstract The alloxan-diabetic rat is characterized by (1) decreased citrate synthase (citrogenase) activity of the liver; (2) a two- to threefold increase in the citrate content of the tissues and (3) a decreased rate of citrate oxidation by the muscle (but not liver). The possible relationship of these changes is discussed.

Effect of insulin on the incorporation of citrate and calcium into the bones of alloxan-diabetic rats

SummaryDiabetes results in osteoporosis, which is characterized by a loss of bone calcium, and thinness of bone shaft. Bone is a major reservoir of calcium and citrate in the body. In this report the effect of diabetes on the deposition of calcium and citrate into rat bones is studied.When [1-5-14C]citrate (50µCi/kg body wt.) was injected intravenously, the bones of untreated alloxan-diabetic rats incorporated less labeled citrate than those of insulin-treated diabetic or control animals. Likewise, when [1-14C]acetate was injected (50µCi/kg body wt.), the labeled citrate synthesized in vivo was incorporated into untreated diabetic rat bones to a significantly lesser extent than in those of insulin-treated diabetic or control rats. In these in vivo studies citrate oxidation was minimized by administration of either an i.v. injection of monofluoroacetic acid (10 mg/kg body wt.) or an i.p. dose of trans-aconitate (2 g/kg body wt.) prior to the administration of labeled acetate or citrate.When45Ca (100µCi/kg body wt.) was injected subcutaneously for 2 consecutive days, the bones of diabetic rats deposited markedly less of the isotope than those of insulin-treated diabetic or control animals.It is concluded that decreased bone mass in diabetes may be a consequence of decreased calcium deposition in the bone.

Studies on Rats with Islet Beta Cell Tumors Induced by Nicotinamide and Streptozotocin

Summary Islet beta cell adenomata were induced in rats by combined treatment with nicotinamide and streptozotocin. Three weeks after treatment marked alterations in glucose tolerance were noted in animals which later exhibited large beta cell tumors. Eight months after treatment, the rats known to have beta cell tumors on the basis of marked hypoglycemia and later confirmed by autopsy showed variable response to a glucose load. Some tumor-bearing rats showed fast response to glucose load, their blood sugar levels were elevated moderately and returned to normal or below normal levels rapidly; these animals are described as having “fast-acting tumors.” Rats with “slow-acting tumors” responded sluggishly to a glucose load; their blood glucose pattern was similar to that of subdiabetic animals. Animals with beta cell tumors exhibited elevated serum insulin levels 30 min after glucose administration. Insulin biosynthesis by beta cell adenomata was demonstrated by in vitro incorporation of [14C]leucine into proinsulin and insulin. In the small number of tumor samples studied, a stimulatory effect of glucose on insulin biosynthesis was observed. Our grateful thanks to Dr. William E. Dulin, The Upjohn Co., Kalamazoo, Michigan, for the gift of streptozotocin, and to Mr. Steven Koziol of Pharmacia Lab, Piscataway, New Jersey, for Phadebas Insulin Test Kits. The Minnesota Medical Foundation aided this research. This paper is dedicated to the memory of the late Dr. Arnold Lazarow.

IMMUNOREACTIVE INSULIN IN RAT SALIVARY GLANDS AND ITS DEPENDENCE ON AGE AND SERUM INSULIN LEVELS

Abstract The major salivary glands of adult rodents contain immunoreactive insulin (IRI). To determine if the concentration of IRI in salivary glands is modulated by the level of serum insulin, insulin immunoreactivity in the parotid and submandibular glands of male rats at different ages (sucklings, pubescent, mature, and elderly) was assayed and compared with corresponding serum insulin concentrations. Salivary glands from suckling rats contained 94 ng/g (wet weight) insulin, which is 1.6 times higher than the level in pubescent rats, and about 10 times higher than levels in mature and elderly rats. No direct relationship between salivary gland content and serum IRI levels was indicated by the data. In an attempt to increase insulin levels in serum, insulin-secreting pancreatic islet adenomas were induced in young male rats by injecting streptozotocin (an Islet tumor-inducing drug) with nicotinamide (which reduces the drugs β-cell cytotoxicity). The mean insulin content of salivary glands from drug-treated rats that had not yet expressed tumors was no higher than controls. After the development of visible tumors of pancreatic islet tissue, however, salivary gland IRI was markerdly elevated, reaching 40 times control levels, whereas serum insulin, and the immunoreactive insulin content of two insulin-sensitive tissues (vis. hepatic, adipose), were elevated only 2-fold. Examination of histologic sections of the parotid and submandibular glands from drug-treated animals revealed no evidence for the formation of salivary tumors. The data indicate that salivary gland insulin content (i) is age-related, being highest in neonates and declining thereafter, (ii) is generally identical in parotid and submandibular glands at a given age, and (iii) is not modulated solely by the animals serum insulin concentration. These results are discussed in regard to the possible sources of insulin detected in the major salivary glands.

Morphometric analysis of the endocrine cell composition of rat pancreas following treatment with streptozotocin and nicotinamide

Using immunohistochemistry and linear scanning, a morphometric analysis was made of the composition of the rat endocrine pancreas at sequential intervals after combined injections of streptozotocin (SZ) and nicotinamide (NA). One week after treatment, the volume of islet tissue was significantly higher than that of the corresponding, saline-injected controls, probably as the result of acute hyperplasia of insulin- and somatostatin-positive cells. However, at all time periods thereafter (6, 20, and 36 weeks), the drug-treated rats showed decreased islet volumes compared to controls. Analysis of aggregate (total) volumes of hormone producing cells at various time periods after drug treatment indicated that decreases in insulin (B-cell) volumes only partially accounted for the observed changes in total islet volume. There were, in addition, early decreases in glucagon (A-cell) and increases in somatostatin (D-cell) volumes. The results suggest that SZ/NA treatment caused limited islet B-cell destruction and transient changes in the proportions of islet A and D cells. Microscopic endocrine tumors were observed at 20 weeks, and both gross and microscopic tumors were observed 36 weeks after SZ/NA treatment. When islet and tumor tissues were included in computation, aggregate volumes of insulin and somatostatin-positive cells were markedly increased, with no significant changes in glucagon-positive cell volumes compared to controls, indicating that the tumors were rich in B and D cells, but poor in A cells. These results are discussed in relation to changes in glucose tolerance and serum insulin levels, and to islet cell volumes following treatment with a diabetogenic dose of streptozotocin alone.

Toxic Effect of Lithium in Mouse Brain

Abstract The effect of lithium ion on glucose oxidation in the cerebrum and cerebellum of mice was measured in vitro by the conversion of isotopic glucose into 14CO2/mg wet weight. Glucose utilization is unaffected by lowest lithium dosage but is inhibited by high lithium concentrations (197–295 mM). Chronic administration of lithium to adult mice decreased the DNA content of the cerebrum and cerebellum at concentrations of 80 and 108 mM. The DNA content of selected postnatal stages of cerebrum and cerebellum was measured starting on Day 1 or 2. This served as another parameter to evaluate glucose oxidation studies at these ages. On the basis of wet weight, both brain parts of neonates of ages 1 and 10 days were approximately one-half that of the adult counterparts. On the basis of DNA content, the cerebrum enhanced its glucose utilization twofold from Day 1 to Day 10 and tripled its utilization from Day 10 to Day 20. The glucose utilization by cerebrum at Day 20 is similar to adult values. In contrast, glucose oxidation in the cerebellum remained relatively constant throughout the postnatal growth. The relative susceptibility of the two brain parts is discussed.