Stimulation of glucose production through hormone secretion and other mechanisms during insulin-induced hypoglycemia.

To assess the role of counterregulatory hormones per se in the response to continuous insulin infusion, overnight-fasted dogs were given 5 mU.kg-1.min-1 insulin intraportally either alone (INS, n = 5), with glucose to maintain euglycemia (INS + GLU, n = 5), or with glucose and hormone replacement [i.e., glucagon, epinephrine, norepinephrine, and cortisol infusions (INS + GLU + HR, n = 6)]. The increases in counterregulatory hormones that occurred during insulin-induced hypoglycemia were simulated in the latter group. In this way, it was possible to separate the effects of hypoglycemia per se from those due to the associated counterregulatory hormone response. Glycogenolysis and gluconeogenesis were measured with a combination of tracer ([ 3-3H]glucose and [U-14C]alanine) and hepatic arteriovenous (AV) difference techniques during a 40-min control and a 180-min experimental period. Insulin levels increased similarly in all groups (to congruent to 250 microU/ml), whereas plasma glucose levels decreased in INS (115 +/- 3 to 41 +/- 3 mg/dl; P less than .05) and rose slightly in both INS + GLU (108 +/- 2 to 115 +/- 4 mg/dl; P less than .05) and INS + GLU + HR (111 +/- 3 to 120 +/- 3 mg/dl; P less than .05) due to glucose infusion. Glucagon, epinephrine, norepinephrine, and cortisol were replaced in INS + GLU + HR so that the increments in their levels were 102 +/- 6, 106 +/- 14, 117 +/- 9, and 124 +/- 37%, respectively, of their increments in INS. At no time was there a significant difference between the hormone levels in INS and INS + GLU + HR. The rise in the counterregulatory hormones per se accounted for only half (53 +/- 9% by the AV difference method and 54 +/- 10% by tracer method) of the glucose production associated with hypoglycemia resulting from insulin infusion. The rate and efficiency of alanine conversion to glucose in the hormone-replacement studies were only 29 +/- 10 and 50 +/- 27% of what occurred during hypoglycemia induced by insulin infusion. In conclusion, the counterregulatory hormones alone (i.e., without accompanying hypoglycemia) can account for only 50% of the glucose production that is present during insulin-induced hypoglycemia. The remaining 50%, therefore, must result from effects of hypoglycemia other than its ability to trigger hormone release.

Norepinephrine and epinephrine in human erythrocyte plasma membranes.

Acetone extract prepared from the plasma membranes of human erythrocytes contained substances which induced the contraction of the thoracic aortic strip of rabbit in vitro and caused blood pressure elevation in rat upon intravenous injection. The contractile response was inhibited by the alpha 1-adrenergic antagonist prazosin. By HPLC/electrochemical detection as well as radioenzymatic assay, large amounts of norepinephrine (NE) (14 +/- 4 [SE] ng/ml packed cells) and epinephrine (E) (16 +/- 2 ng/ml packed cells) were found in the extract. Using the same amounts as in the extract, we were able to demonstrate additive effect between NE and E. The possibility that erythrocyte membranes may play a role in the regulation of NE and E in circulation is suggested.

Role of gluconeogenesis in sustaining glucose production during hypoglycemia caused by continuous insulin infusion in conscious dogs.

The roles of glycogenolysis and gluconeogenesis in sustaining glucose production during insulin-induced hypoglycemia were assessed in overnight-fasted conscious dogs. Insulin was infused intraportally for 3 h at 5 mU.kg-1.min-1 in five animals, and glycogenolysis and gluconeogenesis were measured by using a combination of tracer [( 3-3H]glucose and [U-14C]alanine) and hepatic arteriovenous difference techniques. In response to the elevated insulin level (263 +/- 39 microU/ml), plasma glucose level fell (41 +/- 3 mg/dl), and levels of the counterregulatory hormones glucagon, epinephrine, norepinephrine, and cortisol increased (91 +/- 29 to 271 +/- 55 pg/ml, 83 +/- 26 to 2356 +/- 632 pg/ml, 128 +/- 31 to 596 +/- 81 pg/ml, and 1.5 +/- 0.4 to 11.1 +/- 1.0 micrograms/dl, respectively; for all, P less than .05). Glucose production fell initially and then doubled (3.1 +/- 0.3 to 6.1 +/- 0.5 mg.kg-1.min-1; P less than .05) by 60 min. Net hepatic gluconeogenic precursor uptake increased approximately eightfold by the end of the hypoglycemic period. By the same time, the efficiency with which the liver converted the gluconeogenic precursors to glucose rose twofold. Five control experiments in which euglycemia was maintained by glucose infusion during insulin administration (5.0 mU.kg-1.min-1) provided baseline data. Glycogenolysis accounted for 69-88% of glucose production during the 1st h of hypoglycemia, whereas gluconeogenesis accounted for 48-88% of glucose production during the 3rd h of hypoglycemia. These data suggest that gluconeogenesis is the key process for the normal counterregulatory response to prolonged and marked hypoglycemia.

In vitro phosphorylation of 4-aminobutyrate aminotransferase by cAMP dependent protein kinase.

Highly purified 4-aminobutyrate aminotransferase from pig brain is susceptible to phosphorylation by the purified cAMP-dependent protein kinase catalytic subunit. Up to 0.7 moles of phosphate from ATP-(gamma)-32P can be incorporated per mole of dimeric holoenzyme. Maximum phosphorylation was observed within about 90 minutes at 30 degrees C. Despite the extensive degree of phosphorylation observed, no kinetic property of the enzyme was perceptibly altered. Removal of cofactor had no detectable impact on the extent of phosphorylation but thermal inactivation of the enzyme increased and mild reduction with sodium borohydride decreased the phosphorylatability of the aminotransferase. It was possible to separate the enzyme into phospho and dephospho forms by the use of DEAE chromatography. Validation that the two fractions represented genuine aminotransferase was obtained by proteolytic peptide mapping. The phospho form of the enzyme was found to possess little or no aminotransferase activity while that of the dephospho form exhibited higher specific activity than the purified enzyme prior to phosphorylation. Furthermore, the dephospho form of the enzyme could not be detectably phosphorylated by reincubation with the kinase following DEAE chromatography unless it was subjected to thermal inactivation. The stoichiometry of phosphorylation of the fraction containing 32P from DEAE chromatography was approximately 1 mole/mole of dimer. These results suggest that the substrate for phosphorylation by the kinase is a form of the aminotransferase which is somehow inactivated during routine purification even when extensive precautions are taken to maximally preserve catalytic activity.

Human follicular fluid insulin concentrations.

In mammals, insulin stimulates granulosa cell aromatase activity and steroid production and is a regulating factor of oocyte maturation. To assess the role of insulin in human follicular and oocyte maturation, human follicular fluid was obtained 32-36 h after hCG administration at the time of oocyte recovery for in vitro fertilization. Follicular fluid insulin levels, measured by RIA, ranged from undetectable (less than 2 microU/ml) to 65.4 microU/ml. In women treated with human menopausal gonadotropin (n = 21), clomiphene citrate (n = 4), and human menopausal gonadotropin/clomiphene citrate (n = 14), follicular fluid insulin concentrations were 18.0 +/- 4.3 (+/- SE), 10.2 +/- 4.2, and 12.0 +/- 3.8 microU/ml, respectively (P = NS). Similarly, there was no significant difference in follicular fluid insulin concentrations in follicles with mature (n = 33) or immature (n = 6) oocytes (13.3 +/- 2.7 vs. 24.7 +/- 9.5 microU/ml) or in oocytes which eventually did (n = 35) or did not (n = 4) fertilize (16.4 +/- 3.0 vs. 3.2 +/- 0.8 microU/ml). Follicular fluid insulin levels (n = 30) correlated positively with follicular fluid progesterone levels (P less than 0.05), but not with follicular fluid estradiol or androstenedione levels or the estradiol to androstenedione ratio. The relationship of follicular fluid insulin and progesterone levels suggests that, as in other mammals, follicular fluid insulin may have a physiological role in follicular maturation.

Alpha-adrenergic blockade in vasotonic angina: lack of efficacy of specific alpha 1-receptor blockade with prazosin.

To clarify the possible role of the alpha 1-adrenergic receptor in angina due to coronary artery spasm, a double-blind, randomized, placebo-controlled trial of the specific alpha 1-antagonist, prazosin, was performed. Six patients with vasotonic angina were studied, with efficacy measured by continuous electrocardiographic recording and the tabulation of chest pain and nitroglycerin usage. Despite plasma prazosin levels adequate to produce a six-fold shift in the response to phenylephrine, there was no significant difference in the number of ischemic episodes while taking prazosin (9.8 +/- 6.3 episodes/24 h) compared with placebo (10.5 +/- 6.9). There was also no difference in the length of ischemic episodes, which averaged 231 +/- 35 seconds with placebo and 231 +/- 33 with prazosin. Chest pain and nitroglycerin usage were not altered by prazosin. These data suggest that coronary artery spasm is not primarily caused by an effect on or an abnormality of the coronary vascular alpha 1-receptor.

Tocainide therapy for refractory ventricular arrhythmias.

Tocainide, a congener of lidocaine, was used to treat symptomatic ventricular arrhythmias in 19 patients resistant to or unable to tolerate conventional agents. In this highly selected group, 15 showed good initial responses to oral therapy. Ventricular tachycardia was suppressed to a greater extent than isolated ventricular ectopic depolarizations at any plasma concentration, and upward dose-ranging showed progressive suppression of both. Arrhythmia responsiveness to lidocaine was found to be an excellent predictor of tocainide response. Of the 15 responders, one died 24 hours after stopping therapy, three died while receiving tocainide, nine stopped because of adverse reactions (five allergic), and two continue on therapy at 1 and 4 years. We conclude that tocainide is an effective agent for the short-term suppression of ventricular arrhythmias, particularly ventricular tachycardia sensitive to lidocaine, but a high incidence of adverse effects limits its application to chronic therapy in many patients.

Effects of caffeine on plasma renin activity, catecholamines and blood pressure.

Using a double-blind, randomized, cross-over protocol, we studied the effect of a single dose of oral caffeine on plasma renin activity, catecholamines and cardiovascular control in nine healthy, young, non-coffee drinkers maintained in sodium balance throughout the study period. Caffeine (250 mg) or placebo was administered in a methylxanthine-free beverage to overnight-fasted supine subjects who had had no coffee, tea or cola in the previous three weeks. Caffeine increased plasma renin activity by 57 per cent, plasma norepinephrine by 75 per cent and plasma epinephrine by 207 per cent. Urinary normetanephrine and metanephrine were increased 52 per cent and 100 per cent respectively. Mean blood pressure rose 14/10 mm Hg one hour after caffeine ingestion. There was a slight fall and then a rise in heart rate. Plasma caffeine levels were usually maximal one hour after ingestion but there was considerable individual variation. A 20 per cent increase in respiratory rate correlated well with plasma caffeine levels. Under the conditions of study caffeine was a potent stimulator of plasma renin activity and adrenomedullary secretion. Whether habitual ingestion has similar effects remains to be determined.