Thomas W. Burns

Evidence for Parathyroid Failure in Magnesium Deficiency

Serum immunoreactive parathyroid hormone (IPTH) was low to nondetectable in spite of hypocalcemia in a patient with chronic magnesium deficiency. The administration of magnesium led to parallel increases in serum IPTH, serum calcium, and renal phosphate clearance. These findings support the view that magnesium depletion may result in impaired synthesis or release of parathyroid hormone in man, or both.

Pharmacological Characterizations of Adrenergic Receptors in Human Adipocytes

Three types of adrenergic receptors, beta, alpha-1, and alpha-2, were identified in human adipocytes, isolated from properitoneal adipose tissue, using both the binding of radioactive ligands and the effects of adrenergic agents on receptor-specific biochemical responses. Adrenergic binding studies showed the following results: [(3)H]dihydroalprenolol binding (beta adrenergic) B(max) 280 fmol/mg protein, K(D) 0.38 nM; [(3)H]para-aminoclonidine binding (alpha-2 adrenergic) B(max) 166 fmol/mg protein, K(D) 0.49 nM; [(3)H]WB 4101 binding (alpha-1 adrenergic) B(max) 303 fmol/mg protein, K(D) 0.86 nM. In adipocytes from subcutaneous adipose tissue, [(3)H]dihydroergocryptine binding indicated the presence of alpha-2 but not alpha-1 receptors. Beta and alpha-2 adrenergic receptors appeared to be positively and negatively coupled to adenylate cyclase, respectively. Cells or cell membranes were incubated with epinephrine (10 muM) alone and in combination with the antagonists yohimbine (alpha-2) and prazosin (alpha-1). Epinephrine alone prompted a modest increase in adenylate cyclase activity, cyclic AMP, and glycerol release, an index of lipolysis. Yohimbine (0.1 muM) greatly enhanced these actions whereas prazosin was without effect. The beta agonist, isoproterenol, stimulated glycerol release, whereas the alpha-2 agonist, clonidine, inhibited lipolysis and cyclic AMP accumulation. To assess further alpha-1 receptors, cells were incubated with [(32)P]phosphate and epinephrine (10 muM) alone and in combination with prazosin and yohimbine. Epinephrine alone caused a three- to fourfold increase in (32)P incorporation into phosphatidylinositol. Prazosin (0.1 muM) blocked this action whereas yohimbine (0.1 muM) was without effect. Thus, in a homogeneous cell preparation, the human adipocyte appears to have three different adrenergic receptors, each of which is coupled to a distinct biochemical response.

Myotonic Response Induced by Inhibitors of Cholesterol Biosynthesis

Steroid inhibitors of cholesterogenesis containing nitrogen-substituted side chains induced electromyographic myotonia in rats. Cholesterol reduction or desmosterol accumulation, per se, did not cause myotonia, and cholestrol feeding prevented drug-induced myotonia. Desmosterol accumulation in combination with a specific drug effect may cause the observed myotonia.

ADRENERGIC RECEPTORS AND CYCLIC AMP IN THE REGULATION OF HUMAN ADIPOSE TISSUE LIPOLYSIS

For the past several years our major objective has been the elucidation of mechanisms controlling the release of stored triglyceride by adipose tissue. Since derangement of such mechanisms could well be involved in important human diseases. it was felt that human adipose tissue should be studiedalong with that of appropriate experimental animals-to avoid being misled by species variation. In retrospect, this was a prudent decision since substantial differences between human and rat adipose tissue have been Rat tissue responds to a wide range of lipolytic substances including ACTH, TSH, growth hormone plus dexamethasone, glucagon, and catecholamines. Human adipose tissue is much more selective, responding to catecholamines, TSH, and crude human pituitary fractions associated with TSH, but not responding to ACTH (see FIGURE 1 ) , glucagon, or growth hormone plus dexamethasone. In view of such observations, we felt it important to determine if cyclic AMP played a central role in human adipose tissue lipolysis as it appears to do in the rat. For this purpose, the effects of cyclic AMP, its dibutyryl derivative (DAMP), and theophylline, singly and in combination with insulin, on lipolysis by human and rat adipose tissue have been observed. We have also evaluated the hypothesis that adrenergic receptors may be linked to the cyclic AMP system.x. According to this hypothesis, interaction of a catecholamine with beta receptors causes activation of adenyl cyclase and an increase in cyclic AMP, whereas interaction with alpha receptors reduces the effective concentration of cyclic AMP and leads to an opposite effect on cell function. To evaluate this hypothesis, we have observed the effects of propranolol, a beta blocking agent, and phentolamine, an alpha blocker, on basal and stimulated lipolysis of human and rat cells and tissue fragments. The stimulating substances used were isoproterenol, a relatively pure beta agonist, and epinephrine, a mixed agonist having both alpha and beta stimulating capabilities.

Alpha-2 adrenergic activation inhibits forskolin-stimulated adenylate cyclase activity and lipolysis in human adipocytes

Forskolin at 10 muM caused a 100-fold increase in the intracellular concentration of cyclic AMP and a 6-fold increase in glycerol release in the human adipocyte. These responses are comparable to those prompted by 10 muM isoproterenol. The effects of forskolin on cyclic AMP and lipolysis were dose-dependent. Alpha-2 adrenergic activation, achieved with 10 muM epinephrine and 30 muM propranolol, significantly inhibited forskolin-stimulated cyclic AMP accumulation and glycerol release, shifting the dose-response curves to the right. Forskolin at 10 muM caused a 4.5-fold increase in the adenylate cyclase activity of human adipocyte membranes. When either isoproterenol or epinephrine (0.1 mM) was combined with forskolin, the magnitude of response was substantially greater than the sum of responses achieved by each agent incubated alone.

Observations on Blood Glucose Concentration of Human Subjects During Continuous Sampling

A method for recording blood glucose concentration with use of a continuous sampling technic is described. Sensitivity studies indicate that fluctuations of 3 mg. per 100 ml. or more are reflected on the recording. The major cause of artifact was interruption of flow in the blood sampling tubing or in one of the reagent tubings. Recordings were made during the course of five-hour glucose tolerance tests performed on normal subjects and patients with hyperthyroidism (4), hypothyroidism (1), acute hepatitis (1), and the postgastrectomy syndrome (2). The continuous sampling technic is a practical method to obtain precise temporal and quantitative information relative to fluctuations of the blood glucose concentration under a variety of circumstances.

Tests of a mathematical model of the blood-glucose regulatory system

Abstract A simplified model of the blood-glucose regulatory system has been conformed to data from glucose-tolerance tests in nondiabetics measured by continuous sampling after oral ingestion and by intermittent determinations after different routes of administration. Despite the numerous approximations inherent in the model, all the data were conformed within the limits of experimental error. This extended the applicability of the simplified model used and indicated that the assumption of a single regulatory hormone such as insulin was adequate to fit most normal responses. Small deviations between some of the simulated curves and the actual data suggested modifications that would improve the model. Such changes would sacrifice simplicity for greater accuracy in predicting the detailed response.

Studies on desensitization of adrenergic receptors of human adipocytes

The studies described here were undertaken to determine whether or not desensitization of human adipocyte beta and alpha-2 adrenergic receptors could be demonstrated. Cells, isolated from peritoneal adipose tissue obtained from patients undergoing elective abdominal surgery, were preincubated for 3 hr in buffer alone or in the presence of isoproterenol, 10-5M. Cells in both sets of flasks were then washed and exposed to isoproterenol for 1/2 hr; cyclic AMP was then measured as an end point of beta receptor activation. Cells which had had no prior exposure to isoproterenol responded significantly greater to isoproterenol than did cells that had had prior exposure to the catecholamine, The beta receptor characteristics of cells undergoing beta desensitization were assessed using [3H] dihydroalprenolol. Compared to control cells, adipocytes exposed to isoproterenol had a reduction in Bmax while KD values were the same. Thus desensitization of beta adrenergic receptors of human adipocytes occurs and is associated with down regulation in the number of beta receptors. In comparable studies, preincubation with epinephrine 10-5M did not affect the response of cells to a subsequent exposure to this catecholamine. In alpha-2 receptor binding assays, there was a decreased number of [3H]p-aminoclonidine binding sites, but the level of [3H]yohimbine binding was not altered following the incubation with epinephrine. Thus, desensitization of alpha-2 receptors was not demonstrated.

Insulin Inhibition of Lipolysis of Human Adipocytes: The Role of Cyclic Adenosine Monophosphate

To gain information on the manner in which insulin suppresses lipolysis in man, isolated adipocytes, prepared from subcutaneous adipose tissue, were incubated with insulin (100 μU/ml) alone and in combination with isoproterenol (10−7 M or 10−8 M). Cyclic AMP concentration was measured at 60 min; glycerol release, used as an index of lipolysis, was determined at 45 and 75 min. Insulin consistently reduced both basal and stimulated cyclic AMP and glycerol release: the degree of suppression of each was comparable. In subsequent experiments, the ability of insulin to suppress glycerol release stimulated by isoproterenol, theophylline, and dibutyryl cyclic AMP (dbcAMP), respectively, was compared. Insulin substantially reduced the raised levels of cyclic AMP and glycerol release prompted by isoproterenol and theophylline, but it had little effect on increases caused by dbcAMP. These findings support the view that reduction in cyclic AMP is an important component in the regulation of fat mobilization by insulin.

STUDIES ON THE INTERDEPENDENT EFFECTS OF STRESS AND THE ADRENAL CORTEX ON CARBOHYDRATE METABOLISM IN MAN

It is now well established that the adrenal cortex is involved in some fundamental manner in the metabolic response to injury and illness, but the precise mechanism by which this gland exerts its influence remains clouded. Originally, based on the observations that certain of the metabolic responses to injury do not occur in the absence of the adrenal cortex, and on the evidence of increased secretion of adrenal steroids during stress, it seemed reasonable to interpret the metabolic changes which occur after stress as direct manifestations of hypersecretion of adrenocorticotrophic hormone (ACTH) and adrenocortical steroids (1, 2, 3). The demonstration that overdosage with ACTHor cortisone produced metabolic changes comparable to those after stress made such an interpretation even more attractive. However, as investigation in this area has been pursued, it has become increasingly apparent that the concept described above is an oversimplification and needs modification. Ingle, in a brilliant series of studies, beginning in 1943, has been chiefly responsible for demonstrating, in animals at least, that the adrenal cortex is not directly responsible for the changes under consideration, although adrenocortical hormone must be present for certain of the changes to take place (4, 5, 6, 7, 8). Thus, the adrenalectomized animal exhibits a normal metabolic response to injury if maintained with an amount of adrenocortical hormone which itself does not produce overdosage effects. Under such circumstances, no increase in adrenal activity is possible, and hence adrenocortical hormone may be considered as necessary but not re-