To assess the effects of aging on glucose-mediated glucose disposal and glucose transport, glucose disposal rates were measured in 10 nonelderly (32 +/- 4 yr) and 11 elderly (64 +/- 4 yr) subjects at five different plasma glucose concentrations. Glucose disposal was decreased by 30-35% in the elderly at each level of glycemia (100-350 mg/dl) in the presence of similar levels of hyperinsulinemia (approximately 100 microU/ml), and the 50% effective concentration (EC50) was similar in both the nonelderly (100 +/- 9) and elderly (103 +/- 5 mg/dl). The Michaelis constant (Km) of 3-O-methyl glucose transport in adipocytes was unchanged with aging (3.8 +/- 0.5 vs. 3.2 +/- 0.2 mM) while the maximum velocity of insulin stimulated transport was reduced by 34% in the elderly (8.3 +/- 1.3 vs. 12.6 +/- 1.5 pmol/5 X 10(4) cells per s, P less than 0.05). The insulin resistance of aging is therefore due to a reduction in the capacity of the glucose uptake system, while the affinity of glucose utilization (EC50 and Km) is unchanged. This supports the hypothesis that a reduction in the number of glucose transport and metabolic units occurs with aging, but that each unit functions normally.
R I Fink, P Wallace, J M Olefsky
Feedback regulation of pancreatic enzyme secretion occurs in rats. Whether such a system exists in man remains unsettled and the responsible mechanism is unknown. To investigate this question gastrointestinal intubation and perfusion were performed in 12 healthy subjects. Intraduodenal perfusion of trypsin-inhibited phenylalanine-, oleic acid-, and meal-stimulated chymotrypsin and lipase outputs in a dose-related manner. The minimal concentration of bovine trypsin needed to inhibit pancreatic enzyme secretion was 0.5 g/liter. 1 g/liter caused a maximal suppression of 35 +/- 4% of the phenylalanine-stimulated chymotrypsin release. This inhibitory effect was protease-specific. Intraduodenal perfusion of phenylalanine and oleic acid increased plasma cholecystokinin (CCK) from a basal level of 0.9 +/- 0.06 to 5.3 +/- 0.9 pM and 7.2 +/- 1.3 pM, respectively. Addition of bovine trypsin to the perfusates significantly reduced the plasma CCK level to basal values. This inhibitory effect of trypsin on CCK release was dose dependent and specific to proteases. Therefore, the present studies indicate that feedback regulation of pancreatic enzyme secretion is operative in man and it is mediated by release of CCK.
C Owyang, D S Louie, D Tatum
Initial synthesis of von Willebrand factor (vWf) by cultured human endothelial cells proceeds by formation of a dimer of pro-vWf subunits. These subunits are found only within the cell and have an apparent molecular weight of 240,000-260,000, as measured by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Posttranslational modifications, including proteolytic cleavage, glycosylation, and sulfation, result in the appearance of two additional vWf subunits. The major one migrates with the subunit of plasma vWf at an apparent molecular weight of 220,000-225,000 and the other migrates more slowly than pro-vWf at an apparent molecular weight of 260,000-275,000. These subunits oligomerize to form a set of vWf multimers, which are subsequently secreted into the culture medium. We isolated individual vWf oligomer species from the agarose gel bands and show that vWf minor, or satellite, species differ from major species in subunit composition.
D C Lynch, T S Zimmerman, E H Ling, P J Browning
J L Strominger
We measured alpha 2-adrenoreceptor density as well as affinity for and sensitivity to agonist on intact platelets of normotensive and hypertensive subjects before and after physiological increases in plasma catecholamines. In normotensives, posture-induced rises in plasma catecholamines correlated with reduced alpha 2-adrenoreceptor agonist affinity and fewer high affinity state receptors. Platelet aggregation and inhibition of adenylate cyclase by L-epinephrine also was reduced. Hypertensive subjects had similar rises in plasma catecholamines with upright posture, but showed no change in receptor affinity or sensitivity. No change in platelet alpha 2-adrenoreceptor number occurred in these studies. In vitro incubation with L-epinephrine revealed that platelets from hypertensives had slower desensitization than those from normotensives. Binding studies at different temperatures and with varying sodium concentrations found no thermodynamic or sodium-dependent differences between normotensive and hypertensive groups. These studies demonstrate that platelets from hypertensive subjects exhibit a defect in the ability of physiological concentrations of agonist to desensitize the alpha 2-adrenoreceptor.
A S Hollister, J Onrot, S Lonce, J H Nadeau, D Robertson
The cultured skin fibroblasts from three patients with lacticacidemia were found to have low rates of 1-[14C]pyruvate oxidation in the face of normal pyruvate dehydrogenase activity. After incubation with 1 mM glucose, these three cell strains also exhibited lactate/pyruvate ratios which were three times greater than those of controls. In two of the patients, both ATP and oxygen consumption in fibroblast mitochondrial preparations was deficient with NAD-linked substrates but normal with succinate and ascorbate/N'N'N'N' tetramethyl phenylene diamine. In the third patient, ATP synthesis in mitochondrial preparations was deficient with all substrates tested. Measurement of Rotenone-sensitive NADH-cytochrome c reductase in mitochondrial preparations from skin fibroblasts showed that two of the patients had 14 and 18%, respectively, of control activity. In the third patient, cytochrome oxidase activity was 15% of that in controls. We conclude that respiratory chain defects can be demonstrated in cultured skin fibroblasts with consistency using a number of different techniques.
B H Robinson, J Ward, P Goodyer, A Baudet
Kawasaki syndrome (KS) is characterized by diffuse vasculitis and marked T cell and B cell activation. In this study, sera from 16 patients with acute KS, 15 patients in the convalescent phase of KS, and 19 age-matched controls were assessed for complement dependent cytotoxic activity against 111In-labeled human umbilical vein endothelial (HUVE) cells, Neither sera from patients with KS nor sera from controls had cytotoxic effects on HUVE cells cultivated under standard conditions. Since activated T cells such as those present in acute KS secrete gamma interferon (gamma-IFN), we also examined the effects of sera from acute KS on HUVE cells preincubated with gamma-IFN. We report here that immunoglobulin M (IgM) antibodies in sera from patients with acute KS cause significant (P less than 0.01) killing of gamma-IFN-treated HUVE cells. Pretreatment with interleukin 2, gamma-IFN, or beta-IFN failed to render HUVE susceptible to lysis with acute KS sera. The observed effects were not mediated via immune complexes. The cytotoxic antibodies in acute KS seem to be directed against inducible monomorphic antigenic determinants present on gamma-IFN-treated HUVE cells but not on control or gamma-IFN treated autologous human dermal fibroblasts (HDF). Similarly, acute KS sera also induced lysis of gamma-IFN-treated human saphenous vein endothelial (HSVE) cells but not gamma-IFN treated human saphenous vein smooth muscle (HSVSM) cells. Since gamma-IFN induces the same level of class I and class II major histocompatibility complex (MHC) antigen expression on HDF, HUVE, HSVE, and HSVSM cells, our results suggest that the anti-endothelial cell antibodies in acute KS are directed to gamma-IFN-inducible molecules other than MHC determinants. These observations are further substantiated by the failure of human B cells or monocytes to absorb the anti-endothelial cell activity. Since most vasculitides, including acute KS, are characterized both by marked immune activation and the secretion of lymphokines, antibodies directed to gamma-IFN-inducible endothelial cell antigens may represent a general mechanism for vascular injury.
D Y Leung, T Collins, L A Lapierre, R S Geha, J S Pober
Peripheral vascular effects of opioid peptides are well known, but direct myocardial effects have not been established. We studied the inotropic response of spontaneously beating cultured chick embryo ventricular cells to the enkephalin analogue [D-Ala2]-enkephalin. Amplitude of cell motion increased in a concentration-dependent manner with 0.53 microM [D-Ala2]-enkephalin producing half-maximal response. The mechanism of this positive inotropic effect was investigated by examining alterations in 45Ca influx, cyclic AMP accumulation and adenylate cyclase activity in response to [D-Ala2]-enkephalin. At maximally inotropic concentrations, the 45Ca influx rate increased 39%, adenylate cyclase was stimulated by 30%, and cyclic AMP content rose more than twofold. Thus, in contrast to neural tissue, receptors for enkephalin in cultured heart cells are coupled to adenylate cyclase in a stimulatory manner. Occupancy of these receptors produces an increase in cyclic AMP levels and exerts a positive inotropic effect via a verapamil-sensitive enhancement of Ca influx.
S Laurent, J D Marsh, T W Smith
As the characteristics of sodium and water balance in heart failure remain undefined, we evaluated the hemodynamic, metabolic, and hormonal effects of balanced sodium intake in 10 patients with chronic congestive heart failure. We discontinued diuretics to avoid their confounding influence, and all patients received 1 wk of 10 meq and 100 meq balanced sodium intake and controlled free water. Comparing sodium intake of 10 with 100 meq, the following observations were made. There was weight gain (2.0 kg) and increased sodium excretion (11 +/- 3 to 63 +/- 15 meq/24 h), unaccompanied by increase of blood volume. Both renin-angiotensin system and sympathetic nervous system activity were greater during the 10 meq diet, and suppressed with the 100 meq sodium diet. For both diets, plasma renin and urinary aldosterone excretion were correlated with urinary sodium excretion (r = -0.768, r = -0.726, respectively; P less than 0.005). Systemic hemodynamics were minimally changed with increased sodium intake. However, reversal of vasoconstriction by captopril during the 10 meq diet, and its ineffectiveness during the 100 meq diet, indicated a renin-dependent mechanism in the former, and a renin-independent mechanism in the latter diet. There were two subgroups of response to the 100 meq diet: one group (n = 5) achieved neutral balance, while the second (n = 5) avidly retained sodium and water. Renin-angiotensin system activity was significantly higher in the latter group, and the mechanism for differences in sodium excretion for the subgroups could not be identified by blood volume or hemodynamic parameters. Orthostatic hypotension during tilt was greater during the 10 meq sodium diet, and in all cases, related to ineffective hemodynamic and hormonal compensatory responses.
R J Cody, A B Covit, G L Schaer, J H Laragh, J E Sealey, J Feldschuh
Effects of intraperitoneal injection of allogeneic lymphocytes on insulin secretion were studied in incubated pancreas slices from BALB/c mice. Injection of allogeneic lymphocytes from C57BL/6J (H2b) mice increased insulin secretion, both in basal and 11-mM glucose-stimulated conditions. This effect was only present when at least 5 X 10(6) or 1 X 10(6) cells were injected (in basal and stimulated conditions, respectively). Glucose-induced insulin secretion (3.3-27.5 mM) was significantly increased in pancreata from mice injected with allogeneic lymphocytes. No effect was observed when glucose was not included in the incubation medium. Intraperitoneal injection of Dextran 70 produced no change in glucose-elicited insulin secretion. There were no differences in glucagon and somatostatin (SRIF) secretion obtained from pancreas of mice injected with allogeneic or syngeneic lymphocytes. Injection of allogeneic cells increases insulin secretion (basal and both phases of 11 mM glucose-stimulated secretion). Puromycin significantly inhibited the second phase of insulin secretion. These results suggest that: Injection of allogeneic lymphocytes raises both basal and glucose-stimulated insulin secretion. This effect seems to be connected with the major histocompatibility complex, and to be related to the number of allogeneic cells injected. Injection of allogeneic lymphocytes seems to sensitize the beta cell response to glucose stimulus. Neither glucagon nor SRIF secretion are altered by alloantigen injection. The stimulatory effect of allogeneic lymphocytes is related, at least in part, to insulin synthesis.
J B García, M C Venturino, E Alvarez, L Fabiano de Bruno, M Braun, O H Pivetta, J C Basabe