Johanna A. Pallotta

Effect of Insulin and Glucose Infusions on Sympathetic Nervous System Activity in Normal Man

Recent studies indicate a link between carbohydrate intake and the functional state of the sympathetic nervous system. Fasting or carbohydrate restriction decreases sympathetic activity, while glucose ingestion or dietary supplementation with sucrose increases sympathetic nerve activity. To examine the potential contributions of hyperglycemia and hyperinsulinemia to sympathetic stimulation, sympathetic activity was assessed by measurement of plasma norepinephrine (NE) levels and concomitant cardiovascular indices in nonobese young men during glucose and insulin infusions using glucose clamp techniques. In the insulin infusion studies (euglycemic glucose clamp), insulin was administered at 2 mU/kg/min and 5 mU/kg/min for 2 h while blood glucose was maintained at basal levels by a variable rate of glucose infusion. In the hyperglycemic studies, blood glucose was raised 125 mg/dl above basal and maintained at that level for 2h. In response to both insulin infusions, plasma NE rose progressively over the course of the study, increasing 50% with the 2-mU infusion (from mean basal value of 240 ± 34 pg/ml to 360 ± 41 at 150 min, P < 0.001 for changes over time by analysis of variance) and 117% with the 5-mil infusion (from 254 ± 20 pg/ml to 551 ± 88 at 150 min, P < 0.001). The plasma NE response was greater with the 5-mll than with the 2-mU insulin infusion (P < 0.001), and similarly, was greater during the 2-mU insulin infusion than during a control test in which neither insulin nor glucose was infused (P < 0.001). Associated with the elevations in plasma NE In the 2-mU insulin infusion were increases in pulse rate (P < 0.05), pulse pressure (P < 0.005), and pulse rate - systolic blood pressure product (P < 0.01), and during the 5-mU insulin infusions there were increases in pulse pressure (P < 0.001), mean arterial blood pressure (P < 0.001), and pulse rate - systolic blood pressure product (P < 0.001). Plasma NE did not change during the hyperglycemic glucose clamp nor during control tests, and pulse pressure in the hyperglycemic studies (P < 0.005) was the only cardiovascular measurement increased by these two infusion protocols. The clearance of NE in three subjects was unaffected by the 2-mU insulin infusion. Thus, insulin infusion increases sympathetic nervous system activity in the absence of changes in blood glucose.

Characterization of the insulin resistance of aging.

To clarify the nature of the insulin resistance of aging we studied the dose response for insulin-induced glucose disposal and the binding of insulin to circulating monocytes in healthy young and old men. A total of 49 two-hour euglycemic insulin clamp studies were performed in 17 young and 10 old healthy nonobese subjects. While the old group had lower estimates of lean body mass and greater estimates of total body fat than the young group, these differences did not exceed 5% and did not reach statistical significance. Insulin was infused at 20 mU/m2 per min (young = 8, old = 5); 80 mU/m2 per min (young = 13, old = 9); 200 mU/m2 per min (young = 9, old = 5). Increasing levels of hyperinsulinemia were associated with dose-dependent increases in steady-state glucose infusion rates in young and old. The maximal glucose infusion rates (milligrams per kilogram body weight per minute) were the same for young and old. However, the dose-response curve was shifted to the right in the old subjects. In the four individuals in each age group in whom studies were performed at each dose level, the Km was 54 +/- 14 microU/ml in the young and 113 +/- 11 microU/ml in the old (P less than 0.02). Correction of glucose infusion rate for lean body mass had no effect on comparisons between age groups. These data indicate an age-associated decline in sensitivity of peripheral tissues to insulin without a change in maximal tissue responsiveness. Studies of insulin binding with 14 young and 9 old subjects indicated no effect of age on the insulin binding to receptors on circulating monocytes (young = 5.25 +/- 0.35; old = 6.22 +/- 0.53% of 125I-insulin bound/10(7) cells). These studies suggest that aging may be associated with a postreceptor defect in insulin action manifested by decreased whole-body tissue sensitivity to insulin without a change in tissue responsiveness.

Enhanced plasma norepinephrine response to upright posture and oral glucose administration in elderly human subjects.

Plasma norepinephrine (NE) levels in response to upright posture and to oral glucose ingestion were measured in healthy young and old (greater than 65 yr of age) subjects. Peak plasma NE concentrations with standing were higher in the elderly (1334 +/- 146 pg/ml versus 855 +/- 46; p less than 0.05) and plasma NE remained elevated in the elderly compared with the young subjects even after 15 min of recumbent resting. Following oral glucose plasma NE rose higher in the elderly (79% compared with 32% in the young) and peaked later (120 min after ingestion compared with 60 min in the young). Cardiovascular changes with upright posture and with oral glucose were similar in young and old. Alterations in disappearance of NE from the circulation could not account for the greater elevations in plasma NE concentration in the elderly either, since the rates of fall in circulating NE levels following termination of an NE infusion were the same in both groups. The metabolic clearance rate of NE was unchanged. Thus, the plasma NE responses to stimulation by standing and by oral glucose ingestion are enhanced in elderly subjects.

Regulation of parathyroid hormone release and cytosolic calcium by extracellular calcium in dispersed and cultured bovine and pathological human parathyroid cells.

Alterations in parathyroid glandular sensitivity to calcium may contribute to the hypersecretion of PTH in hyperparathyroidism. Since the cytosolic calcium concentration may mediate the effects of extracellular calcium on PTH release, we have employed the calcium-sensitive intracellular dye QUIN-2 to examine the relationship between extracellular calcium, cytosolic calcium, and PTH secretion in adult, neonatal, and cultured bovine as well as pathological human parathyroid cells. PTH release was measured using C- and N-terminal radioimmunoassays. Neonatal bovine parathyroid cells showed a greater set-point for secretion (the Ca++ concentration causing half of the maximal inhibition of PTH release) than adult cells (1.27 +/- 0.11 vs. 1.06 +/- 0.11 mM extracellular calcium, P less than 0.01), and a slightly higher extracellular calcium was necessary to raise the cytosolic calcium concentration to a given level in neonatal than in adult bovine parathyroid cells. In individual neonatal and adult cell preparations, there was a close correlation between the set-point for secretion and the set-point for cytosolic calcium (r = 0.832, P less than 0.001). In cells from five human parathyroid adenomas, which had an increase in set-point for secretion, the extracellular calcium concentration necessary to raise the cytosolic calcium concentration to a given level was slightly greater than in the neonatal cells. In four preparations of human parathyroid cells there was a significant correlation between the set-points for secretion and cytosolic calcium (r = 0.856, P less than 0.01). Because neonatal bovine and pathological human parathyroid glands show cellular hyperplasia, we studied the temporal relationship between cellular proliferation and the regulation of PTH release and cytosolic calcium concentration in cultured bovine parathyroid cells. Cellular proliferation, estimated by 3H-thymidine incorporation, increased significantly in culture from 104 +/- 10.1 counts/well on day 1 (first 24 h in culture) to 588 +/- 188 and 6,156 +/- 649 counts/well on days 2 and 4, respectively. In cultured cells on day 1, highly Ca++ (2-3 mM) inhibited maximal PTH release by 58.8 +/- 3.2%, which decreased significantly (P less than 0.001) to 38.2 +/- 1.9 and 17.1 +/- 3.7% on days 2 and 4, respectively. The cytosolic calcium observed at 3 mM calcium on day 1 was 701 +/- 43 nM, which declined to 466 +/- 60 and 314 +/- 14 nM on days 2 and 4 (P less than 0.05). There was a close correlation between this progressive decrease in maximal inhibition of PTH release and the cytosolic calcium at high extracellular calcium in cultured cells (r = 0.99, P < 0.001). Thus, during active proliferation of cultured cells, there is an alteration in the regulation of cytosolic calcium at a given extracellular calcium concentration, and changes in the regulation of PTH release and cytosolic calcium by extracellular calcium may be related to enhanced cellular proliferation.

Clinical Studies of Alcoholic Ketoacidosis

Seven episodes of severe ketoacidosis in six nondiabetic patients were recognized at this hospital within an eighteen month period. All were women; one pregnant patient experienced two episodes at twenty-eight and thirty-two weeks gestation. All patients admitted to heavy chronic alcohol intake and drinking binges. On admission, these patients were conscious and alert. Mean values were 143 mg./100 ml. for plasma glucose and 7.25 for arterial pH. Plasma bicarbonate was depressed with a mean anion gap of 18. Beta-hydroxybutyrate/acetoacetate ratio averaged 5.2. All patients had liver function abnormalities. Mean serum immunoreactive insulin was low, 5μU./ml. (n=2), while cortisol was markedly elevated at 76.5μg. per cent (n=3); mean growth hormone level was 14.1 ng./ml. (n=3). Free fatty acid concentration, measured on admission in one episode, was 1,945 mEq./L. Therapy with glucose, saline, and minimal amounts of alkali led to prompt recovery. Circulating levels of cortisol, insulin and growth hormone were measured serially in one patient during recovery; they quickly returned to normal. The dissociation of severe ketosis from glycosuria and hyperglycemia in these patients raises important questions concerning coupling of ketogenesis to gluconeogenesis. The striking preponderance of women, including one pregnant patient, reported with this condition also suggests a possible role for ovarian and placental hormones in its pathogenesis; fetal drain on carbohydrate reserves may further contribute to the tendency to ketosis. Alcoholic ketoacidosis may be relatively common, since we saw one case of this syndrome for about every four of diabetic ketoacidosis during this period.

Influence of Age on Clearance of Insulin in Man

With advancing age, glucose-induced insulin release is decreased in vitro, yet circulating insulin levels after glucose challenge are not decreased in the elderly. Age-related changes in insulin clearance may contribute to this discrepancy. We employed the 2-h euglycemic clamp technique to examine insulin clearance (C1) during steady-state insulin infusions (N = 53) at rates of 20, 80, and 200 mU/m2/min in healthy young (N = 16, age range 22–37 yr, relative weight 1.07 ± 0.03) and old (N = 10, age range 63–77 yr, relative weight 1.14 ± 0.03) men. Steady-state insulin levels were statistically significantly higher (P < 0.01) in the elderly at each infusion rate. C1 was 40% lower (p < 0.01) in the infusion rate. There was no effect of increasing relative weight on C1 within age groups. Within each age group, C1 was similar in insulin infusion rates of 20 and 80 mU/m2/min (young P < 0.05, old P < 0.001), implying a saturable system for insulin clearance. Alterations in C1 contribute to changes in insulin levels with age and may reconcile the discrepancy between in vivo and in vitro studies of glucose-induced insulin release. These results indicate the value of evaluating C1 as one determinant of circulating insulin level in states of abnormal insulin physiology.

Impaired in Vivo Insulin Clearance in Patients With Severe Target-Cell Resistance to Insulin

The concentration of insulin in plasma is determined by both its rate of secretion and its rate of clearance from the plasma compartment. The effect of marked insulin resistance on insulin clearance in vivo has not been determined in man. We have employed the euglycemic insulin clamp technique to measure insulin sensitivity and insulin clearance in 16 control subjects and in 4 subjects with marked target-cell resistance to insulin. Two insulin-resistant patients had reduced receptor concentration on peripheral mononuclear cells, and two patients had normal receptor number and affinity. During 80-mU/m2/min insulin clamp studies, the clearance rate in each insulin-resistant patient was lower than that in any controls; the mean insulin clearance rate was 511 ± 74 ml/m2/min in control subjects and 205 ml/m2/min (P < 0.001) in insulin-resistant patients. These findings demonstrate an association between marked target-cell resistance to insulin and impaired in vivo insulin clearance, and suggest an important role for receptor-mediated pathways in insulin clearance in vivo.

Response of plasma insulin and growth hormone to carbohydrate and protein feeding

Abstract Twelve male patients were fed diets of glucose, protein, starch and combinations of protein with either glucose or starch. Protein was a weak stimulus to insulin production compared to carbohydrate, which is a powerful insulin secretagogue. Combination feedings led to a synergistic effect on insulin secretion. Protein caused a greater plasma insulin response in the two patients with maturity-onset diabetes than it did in non-diabetic subjects. Protein feeding led to a rise in plasma HGH at the 2nd to the 4th hour. After glucose or starch there was a rise in plasma HGH at the 4th–5th hour following an initial suppression. HGH responses were blunted when combinations of carbohydrate and protein were fed. These interrelationships are discussed.

Effect of insulin on plasma norepinephrine and 3,4-dihydroxyphenylalanine in obese men

Increasing evidence relates serum insulin level and blood pressure in obese individuals. Although the connection between these two factors is not established, a common presumption is that the sympathetic nervous system is somehow involved, in part, because laboratory studies demonstrate insulin stimulation of sympathetic and cardiovascular activity. Because the obese may exhibit altered responsiveness to insulin action, the current investigation compared cardiovascular and neurohumoral responses to euglycemic insulin infusion (200 mU/m2/min) in obese and lean men. At baseline, obese men displayed higher glucose and insulin levels, faster pulse rates, and elevated mean arterial pressures (MAP) than lean controls; plasma norepinephrine (NE) and 3,4-dihydroxyphenylalanine (DOPA) concentrations, however, did not differ. During insulin infusion, pulse rate increased equally in obese and lean subjects (from 69 to 78 min-1 in obese and from 56 to 66 min-1 in lean subjects), while MAP remained unchanged in both groups. Elevations in plasma NE (+85 pg/mL in obese and +109 in lean pg/mL) and reductions in plasma DOPA (-233 pg/mL in obese and -376 pg/mL in lean) did not differ between groups. Sodium excretory rate decreased during insulin infusion in lean subjects by 2.2 mEq/h but increased in obese by 5.3 mEq/h (difference in response between groups, P = .024). Thus, in these obese men, cardiovascular and sympathetic responses to insulin persist despite evidence of moderate insulin resistance; increased sympathetic activity, as a cause for the resting tachycardia and borderline hypertension at baseline, seems unlikely.

Selective arteriography, venography and venous hormone assay in diagnosis and localization of parathyroid lesions☆☆☆

Abstract The results of parathyroid arteriography, venography and venous hormone assay were evaluated in 50 patients with proved hyperparathyroidism. Complete selective venous sampling representative of drainage sites in both upper and lower poles of the thyroid lobes was obtained in 94 per cent of the patients and was found to be an innocuous technic. Venous sampling alone provided correct localization and differentiation of parathyroid lesions in all patients without prior exploration. Venous sampling followed by limited arteriography provided specific identification of abnormal solitary parathyroid glands in 17 of 20 patients with unsuccessful prior explorations. Selective-magnification thyroid arteriography was positive in all patients retrospectively, but only a 77 per cent prospective accuracy could be achieved because of difficulty in differentiating thyroid lesions from parathyroid lesions. Venography without hormone assay was only occasionally helpful. These technics provided a significant contribution to the management of the previously explored patients. Furthermore, selective venous hormone assay should be considered in the routine preoperative evaluation of patients with primary hyperparathyroidism.