David M. Kipnis

Measurement of “True” Glucose Production Rates in Infancy and Childhood with 6,6-Dideuteroglucose

“New” glucose production has been measured in 54 infants and children for the first time by continuous three-to-four-hour infusion of the safe, nonradioactive tracer 6,6-dideuterogiucose. The use of combined gas chromatography-mass spectrometry with monitoring of selected ions allowed deuterium enrichment in blood glucose to be measured on microliter samples with an error of less than 2 per cent. In the young child, glucose production increased in a slightly curvilinear manner from 1 kg. to 25 kg. body weight, when it reached 140 mg. per minute, almost the adult value of 173 mg. per minute (2.28 ± 0.23 mg./kg. ·min., mean ± S.E.). Normalized for weight, glucose production in premature infants was 5.46 ± 0.31 mg./kg. ·min., in term neonates averaged 6.07 ± 0.46 mg./kg. · min., in children below the age of six years was 7.1 ± 0.27 mg./kg.· min., and in late childhood averaged 5.4 ± 0.28 mg./ kg.· min. Relative to estimated brain weight, however, glucose production was essentially linear from the 1-kg. premature infant to the 80-kg. adult. These data, the first measurements of “new” glucose production in childhood, suggest that brain size may be a principal determinant of those factors that regulate hepatic glucose output throughout life.

The role of adrenergic mechanisms in the substrate and hormonal response to insulin-induced hypoglycemia in man.

Sequential determinations of glucose outflow and inflow, and rates of gluconeogenesis from alanine, before, during and after insulin-induced hypoglycemia were obtained in relation to alterations in circulating epinephrine, norepinephrine, glucagon, cortisol, and growth hormone in six normal subjects. Insulin decreased the mean (+/-SEM) plasma glucose from 89+/-3 to 39+/-2 mg/dl 25 min after injection, but this decline ceased despite serum insulin levels of 153+/-22 mul/ml. Before insulin, glucose inflow and outflow were constant averaging 125.3+/-7.1 mg/kg per h. 15 min after insulin, mean glucose outflow increased threefold, but then decreased at 25 min, reaching a rate 15% less than the preinsulin rate. Glucose inflow decreased 80% 15 min after insulin, but increased at 25 min, reaching a maximum of twice the basal rate. Gluconeogenesis from alanine decreased 68% 15 min after insulin, but returned to preinsulin rates at 25 min, and remained constant for the next 25 min, after which it increased linearly. A fourfold increase in mean plasma epinephrine was found 20 min after insulin, with maximal levels 50 times basal. Plasma norepinephrine concentrations first increased significantly at 25 min after insulin, whereas significantly increased levels of cortisol and glucagon occurred at 30 min, and growth hormone at 40 min after insulin. Thus, insulin-induced hypoglycemia in man results from both a decrease in glucose production and an increase in glucose utilization. Accelerated glycogenolysis produced much of the initial, posthypoglycemic increment in glucose production. The contribution of glycogenolysis decreased with time, while that of gluconeogenesis from alanine increased. Of the hormones studied, only the increments in plasma catecholamines preceded or coincided with the measured increase in glucose production after hypoglycemia. It therefore seems probable that adrenergic mechanisms play a major role in the initiation of counter-regulatory responses to insulin-induced hypoglycemia in man.

Plasma Insulin Responses to Glucose and Tolbutamide of Normal Weight and Obese Diabetic and Nondiabetic Subjects

Plasma insulin responses to oral glucose (100 gm.) and intravenous tolbutamide (1.0 gm.) before and after dexamethasone were determined in normal, obese nondiabetic, normal-weight diabetic and obese diabetic subjects. Fasting plasma insulin levels were significantly higher in obese nondiabetic and obese diabetic subjects than in normalweight nondiabetic and diabetic individuals. Total plasma insulin response to oral glucose in obese nondiabetics was threefold greater, in obese diabetic 2.2-fold greater, and in normal-weight diabetics 1.7-fold greater than that observed in normal individuals. When the plasma insulin responses of obese diabetic and nondiabetic subjects were measured at comparable blood sugar levellN insulin secretion was demonstrated to be significantly impaired in the diabetic group. The initial plasma insulin response (i.e., thirty-minute level) to glucose of diabetic subjects is markedly impaired when compared to normal-weight and obese nondiabetic individuals. Total plasma insulin response to intravenous tolbutamide was fourfold greater in obese nondiabetic and obese diabetic subjects than in normal-weight nondiabetic and diabetic individuals. Following dexamethasone (2 mg. q.i.d. × 2), plasma insulin response to intravenous tolbutamide was increased fivefold in normal subjects, threefold in obese nondiabetics, 1.6-fold in obese diabetics and twofold in nonobese diabetics. The results of these studies indicate that obesity is associated with a hypersecretory insulin response to both tolbutamide and glucose. The presence of overt diabetes melitus indicates an impairment in insulin response in both obese and nonobese individuals when compared to their appropriate nondiabetic controls. Dexamethasone provokes a marked compensatory insulin response in nondiabetics and makes more apparent the impaired secretory capacity of diabetic subjects. Maturity-onset diabetes in nonobaese individuals appears to be a consequence primarily of insulin deficiency. In obese diabetic individuals, however, even though secretory capacity may be significantly greater than observed in nonobese diabetics, impaired carbohydrate tolerance develops because insulin antagonism associated with obesity per se.

The effect of oxotremorine and atropine on cGMP and cAMP levels in mouse cerebral cortex and cerebellum

Abstract Guanosine 3′, 5′ cyclic monophosphate (cGMP) and adenosine 3′,5′ cyclic monophosphate (cAMP) were measured in the cerebellum and cerebral cortex of mice after treatment with oxotremorine and atropine. Oxotremorine treatment results within 3–5 minutes in a 70% increase in cGMP in cerebral cortex and cerebellum. Pretreatment with atropine prevents the oxotremorine induced rise in cGMP. Following treatment with atropine alone, cGMP levels rise in cerebral cortex and fall in cerebellum. Atropine has no effect on cAMP levels, but oxotremorine decreases cAMP levels in both cerebral cortex and cerebellum. These results are interpreted as indicating that 1) cGMP is involved in cholinergic neurotransmission, and 2) cGMP and cAMP have idependent functional roles in the central nervous system.

Functional heterogeneity of the intracellular amino acid pool in mammalian cells

Abstract 1. 1. The kinetics of tracer amino acid appearance in the intracellular amino acid pool and in cellular protein was examined under steady-state conditions in the intact rat-diaphragm preparation and in isolated guinea pig and rabbit-lymph node cells. 2. 2. The concentration ratio of tracer amino acid between the extracellular and intracellular amino acid pools approached equilibrium exponentially whereas incorporation into cellular protein proceeded linearly from the earliest time period studied. 3. 3. A theoretical formulation is presented predicting the kinetics of amino acid incorporation into cellular protein if the total intracellular amino acid pool were an obligatory intermediate in protein synthesis. 4. 4. The marked deviation of experimental observations from those theoretically predicted has been interpreted to indicate functional heterogeneity (compartmentation) of the intracellular amino acid pool.

Secretion of various endocrine substances by ACTH-secreting tumors--gastrin, melanotropin, norepinephrine, serotonin, parathormone, vasopressin, glucagon.

Six cases of ACTH‐secreting tumors—which are shown to secrete additional hormonal substances—are reported. The high incidence of this phenomenon among our 15 observed cases with ACTH‐secreting tumor suggests that poly‐humoral secretion in ACTH‐secreting tumors is more common than previous reports would indicate. Although several of our cases are unique in that the particular combination of endocrine substances produced has not been previously reported, the tumor types (islet‐cell carcinoma, carcinoid, pheochromocytoma, oat‐cell carcinoma) are in general those which may be endocrine active in a variety of ways. Ectopic corticotrophin production arises principally in tumors of endocrine tissue, neuroendocrine tissue, endocrine dependent tissue and in oat‐cell carcinoma of the thorax. The possibility that oat‐cell carcinoma of the lung and mediastinum may be a more malignant variety of carcinoid tumor is not proven but should not yet be discounted.

Effects of amphetamine, chlorpromazine and reserpine on cyclic GMP and cyclic AMP levels in mouse cerebellum

Abstract Cyclic GMP and cyclic AMP levels were measured in the cerebella of mice treated with several drugs which affect CNS function. D-amphetamine produced a two fold increase in cyclic GMP levels, whereas chlorpromazine and reserpine reduced cyclic GMP levels 62% and 73%, respectively. The effect of d-amphetamine on cyclic GMP levels was blocked by chlorpromazine, but not by atropine. Chlorpromazine did not block the increase in cyclic GMP levels which follows oxotremorine treatment. None of the above agents altered cyclic AMP levels. These results offer further support for the concept that cyclic GMP is involved in neural function and is controlled by mechanisms independent of those regulating cyclic AMP metabolism.

Hypoalaninemia: a concomitant of ketotic hypoglycemia

The cause of of ketotic hypoglycemia, the commonest form of hypoglycemia in childhood, is not known. The present study was undertaken to determine whether the primary defect in this condition is a deficiency of gluconeogenic precursor(s) or an abnormality in the hepatic gluconeogenic enzyme system. Plasma glucose, alanine, and insulin and blood beta-hydroxybutyrate (beta-OHB), pyruvate, and lactate levels were determined in eight ketotic hypoglycemic children and seven agematched controls maintained on a normal diet and after being fed a provocative hypocaloric low-carbohydrate diet (1200 kcal/1.73 m(2), 15% carbohydrate, 17% protein, and 68% fat). On a normal diet, overnight fasting plasma alanine (211+/-10 muM) and glucose (68+/-4 mg/100 ml) were significantly lower and blood beta-OHB (1.22+/-0.37 mM) significantly higher in ketotic hypoglycemic children than in controls (alanine, 315+/-15 muM; glucose, 81+/-3 mg/100 ml; beta-OHB, 0.18+/-0.08 mM). All ketotic hypoglycemic children developed symptomatic hypoglycemia (33+/-3 mg/100 ml) and ketosis (beta-OHB, 3.70+/-0.32 mM) 8-16 hr after starting the provocative diet and these changes were associated with a further decline in plasma alanine (155+/-17 muM). Normal children, even after 36 hr on this diet, maintained higher plasma glucose (48+/-2 mg/100 ml) and alanine (225+/-5 muM) and lower beta-OHB levels (2.56+/-0.44 mM). Intravenous infusions of alanine (250 mg/kg) uniformly restored the hypoglycemic plasma glucose levels of ketotic hypoglycemic children to normal. Cortisone acetate (300 mg/m(2)), given orally in three divided doses during feeding of the provocative diet, produced a 3- to 4-fold increase in plasma alanine within 4-6 hr after beginning steroid therapy and completely prevented the development of hypoglycemia and ketosis. Quantitative amino acid profiles were performed and demonstrated that alanine was the only gluconeogenic amino acid which differed significantly between the two groups. Plasma insulin and blood lactate and pyruvate levels did not differ significantly between normal and ketotic hypoglycemic children under all conditions examined. These results support the hypothesis that a deficiency in gluconeogenic precursor (e.g., alanine) rather than a defect in the hepatic gluconeogenic enzyme apparatus represents the most likely factor in the pathogenesis of ketotic hypoglycemia.

A new colchicine binding assay for tubulin

Abstract A simple and sensitive procedure is described for assaying tubulin, the subunit protein of microtubules. Tubulin is equilibrated with [3H]colchicine after which the remaining free colchicine is removed by adsorption to activated charcoal. Values for the amount of tubulin in various rat tissues have been determined by both the charcoal method and by gel filtration on Sephadex G-100, and in all cases the values determined by the two methods correspond closely.

Juvenile diabetes mellitus, a deficiency in insulin.

The plasma insulin, growth hormone, nonesterified fatty acids and glucose responses to the oral ingestion of glucose, the intravenous administration of tolbutamide and the infusion of arginine were studied in fourteen newly diagnosed juvenile diabetic children. Fasting plasma insulin levels did not differ significantly between diabetic and normal children, although the mean fasting blood glucose level of the diabetic children was threefold greater than that of the normal subjects No detectable plasma insulin response was observed in the diabetic subjects during all three tests. Fasting plasma growth hormone levels and plasma growth hormone responses were similar in both normal and diabetic children. The fasting free fatty acid level of the diabetic children was significantly higher than in normal children, but fell during all three tests. With use of a variety of sensitive procedures, no antibodies to human insulin were demonstrable in the sera of newly diagnosed juvenile diabetics.