Gerich, John E.

Ketone bodies have been suggested to have a proteinsparing effect, since infusion of Na-β-hydroxybutyrate in man decreases plasma alanine concentrations and urinary nitrogen (N) excretion. To test this hypothesis, six normal postabsorptive volunteers were infused with Na-β-hydroxybutyrate for 3 h. Rates of glucose, leucine carbon, and alanine appearance and disappearance from the plasma space were traced with [3-3H]glucose, L-[6,6,6-2H3]leucine, and [2,3,3,3-2H4]alanine. Rates of leucine N appearance and disappearance and the rate of transfer of leucine N to alanine were assessed with [15N]leucine. During ketone body infusion, plasma alanine decreased (P < 0.05), whereas plasma leucine increased (P < 0.05). Rates of alanine appearance increased (5.3 ± 0.3 to 7.8 ± 0.6 μmol/kg · min), but the increase in its rate of disappearance was sightly greater, accounting for the decrease in plasma alanine concentration. Leucine N flux and the rate and percent of leucine N transferred to alanine increased, whereas leucine carbon flux was unchanged. To determine the effect of the alkalemia induced by Na-β-hydroxybutyrate, four additional subjects were infused with NaHCO3. Alkalemia had no effect on leucine N or carbon flux or on the rate of appearance of alanine, but increased the rate of alanine disappearance, resulting in a decrease in the plasma alanine concentration. Since the rate of appearance of leucine carbon was unaltered during the infusion of Na-β-hydroxybutyrate, it is unlikely that hyperketonemia per se decreases proteolysis in postabsorptive man.

To evaluate the roles of counterregulatory hormones and insulin antibodies in the impairment of plasma glucose recovery from hypoglycemia in diabetes mellitus, and to assess the relationship between the glucagon response and duration of the disease, 21 insulin-dependent diabetic patients and 10 nondiabetic subjects were studied. The diabetics consisted of 5 patients with recent onset of diabetes (< 1 mo); 11 with 2.6 ± 0.3 (mean ± SEM) yr duration of diabetes, 5 of whom had insulin antibodies; and 5 patients with long-term diabetes (21 ± 3 yr), insulin antibodies, and autonomic neuropathy. During insulin-induced hypoglycemia (28 mU/m2 × min for 60 min) in patients with recent-onset diabetes, plasma free insulin, glucose, and counterregulatory hormone concentrations did not differ from those of nondiabetic subjects. In patients with insulin antibodies, the disappearance of insulin after insulin infusion was delayed, and both restitution of normoglycemia and plasma glucagon response were blunted compared with patients without antibodies. When glucagon was infused (80–130 ng/m2 × min) during hypoglycemia in diabetics with impaired glucagon responses in order to simulate normal glucagon responses, plasma glucose recovery was normalized in patients without antibodies but not in those with antibodies. In patients with long-standing diabetes, restitution of normoglycemia was further impaired and this was associated with an absent plasma glucagon response and a diminished plasma epinephrine response. Plasma glucagon responses to hypoglycemia were inversely correlated to the duration of diabetes (r = −0.943; P < 0.0005). It is concluded that impaired A-cell secretion is the predominant mechanism for the delayed glucose recovery after hypoglycemia in diabetic patients without insulin antibodies and normal epinephrine responses. Slowed disappearance of insulin due to the presence of insulin antibodies further delays the restoration of normoglycemia. Patients with long-standing diabetes and autonomic neuropathy exhibit decreased epinephrine secretion, which leads to an additional retardation of glucose recovery. Since plasma glucagon and epinephrine responses to hypoglycemia were normal at the onset of diabetes but diminished in long-term diabetes, it appears that the impaired glucagon and epinephrine responses to hypoglycemia are acquired defects that develop subsequent to B-cell failure.

In order to assess the adrenergic contribution to hypoglycemie glucose counterregulation in type | diabetes mellitus and to determine whether the adrenergic contribution is mediated through beta1- or beta2-adrenergic receptors, hypoglycemia was induced by an i.v. insulin infusion (30 mU/m2 · min) for 60 min in 11 insulin-dependent diabetic patients (IDDM), 5 with normal plasma glucagon responses and 6 with blunted responses, and also in 7 age-weight-matched nondiabetic subjects. Rates of plasma glucose decrease and postnadir increase, as well as plasma concentrations of free insulin and of counterregulatory hormones, were measured when insulin was infused alone, and when insulin was infused along with propranolol (a beta1- and beta2-adrenergic receptor antagonist) or metoprolol (a selective beta1-antagonist). Postnadir plasma glucose recovery was decreased in IDDM with blunted plasma glucagon responses (21 ± 0.8 µmol. L−1 min−1, P < 0.001), but was normal in patients with normal plasma glucagon responses (30 ± 0.4 versus 33 ± 0.5 μmol · L−1 · min−1 in nondiabetic subjects, P = NS). Postnadir plasma glucose recovery was not affected by either propranolol or metoprolol in normal subjects and in IDDM with normal glucagon responses. However, in IDDM with blunted plasma glucagon responses, postnadir plasma glucose recovery was further decreased by propranolol (14 ± 0.6 µmol. L−1 min1, P < 0.01), but was unaffected by metoprolol (22 ± 0.9 μmol. L 1 min1, P = NS). These results demonstrate that, as in normal man, adrenergic mechanisms are not essential for normal plasma glucose recovery from hypoglycemia in IDDM with normal plasma glucagon responses. However, in IDDM with blunted plasma glucagon responses, adrenergic mechanisms become important for glucose counterregulation although they do not fully compensate for the blunted plasma glucagon responses. Since propranolol delayed plasma glucose recovery from hypoglycemia in IDDM with blunted glucagon responses but metoprolol had no effect, the adrenergic contribution to glucose counterregulation appears to be mediated through beta2-adrenergic receptors.

The present studies were undertaken to assess the mechanisms responsible for growth hormone-induced insulin resistance in man. The insulin dose-response characteristics for suppression of glucose production and stimulation of glucose utilization and their relationship to monocyte insulin binding were determined in six normal volunteers after 12-h infusion of growth hormone and 12-h infusion of saline. The infusion of growth hormone (2 μg · kg−1 · h−1) increased plasma growth hormone nearly threefold (to ≃9 ng/ml) within the range observed during sleep and exercise. This increased plasma insulin (14 ± 1 versus 8 ± 1 μU/ml, P < 0.005) concentrations without significantly altering plasma glucose concentrations or basal rates of glucose production and utilization. Insulin dose-response curves for both suppression of glucose production (half-maximal response at 37 ± 3 versus 20 ± 3 μu/ml, P < 0.01) and stimulation of glucose utilization (half-maximal response at 98 ± 8 versus 52 ± 8 μU/ml, P / 0.01) were shifted to the right with preservation of normal maximal responses to insulin. Monocyte insulin binding was unaffected. Thus, except at near maximal insulin receptor occupancy, the action of insulin on glucose production and utilization per number of monocyte insulin receptors occupied was decreased. These results indicate that increases in plasma growth hormone within the physiologic range can cause insulin resistance in man, which is due to decreases in both hepatic and extrahepatic effects of insulin. Assuming that insulin binding to monocytes reflects insulin binding in insulin sensitive tissues, this decrease in insulin action can be explained on the basis of a postreceptor defect.

The optimal route for insulin administration by insulin infusion devices has not been established. To assess the differences between the peripheral venous and portal venous routes of insulin administration on postprandial metabolic responses, six alloxan-diabetic dogs were studied on four occasions. On the first, the insulin infusion rates given by the Biostator for disposal of a mixed meal were recorded electronically. On two subsequent occasions, these insulin infusion profiles were administered by a peripheral vein. On an additional occasion, the identical insulin infusion profile was given via the portal vein. No differences were observed in basal or peak plasma glucose, insulin, glucagon, branched chain amino acids, and lactate concentrations between portal and peripheral venous insulin administration. Furthermore, no differences in isotopically ([2-3H]glucose) determined rates of systemic glucose appearance and disappearance were observed between the two routes. Although preprandial plasma alanine concentrations were greater when insulin was infused via the portal vein, the postprandial increments did not differ from those observed during infusion of insulin by a peripheral vein. These studies suggest that under the current experimental conditions, there appears to be no difference in the disposition of a mixed meal, measured as net whole body glucose appearance and disappearance rates, when insulin is administered in an open-loop fashion via either a peripheral or the portal vein.

Human plasma somatostatin-like immunoreactivity (SLI) is heterogeneous. Moreover, human plasma contains a somatostatin-binding protein, enzymes that degrade the peptide, and other poorly characterized substances that interfere in radioimmunoassays of the peptide. Previously described extraction methods only partially circumvent these problems and generally result in less than adequate recovery. The present studies were therefore undertaken to determine whether gel filtration of plasma could be used to avoid these difficulties and provide a means for radioimmunoassay of plasma somatostatin in man. Gel filtration (Sephadex G25) of human plasma and subsequent radioimmunoassay of column eluates using five different antisera uniformly demonstrated two fractions of SLI in plasma. One fraction (peak I) eluted in the void volume and had an apparent molecular weight in excess of 150,000 daltons; this material comprised most of the plasma SLI, did not dilute in parallel with synthetic somatostatin, and decreased immunoprecipitability of both N-tyrosine- and tyrosine-11-125I somatostatin. The second fraction (peak II) coeluted with synthetic somatostatin (apparent mol. wt. 1600 daltons), diluted in parallel with synthetic somatostatin, and did not appreciably degrade either 125I-tyrosylated analogue. Synthetic somatostatin added to plasma was recovered nearly completely (95 ± 3%) in peak II SLI. Infused synthetic somatostatin increased peak II SLI but did not alter peak I SLI. These results demonstrate that gel chromatography of human plasma before assay can be used to circumvent problems of immunoheterogeneity, 0degradation, and nonspecific interference while providing adequate recovery. With this method, mean plasma somatostatin concentrations (peak II SLI) in postabsorptive normal volunteers ranged from approximately 40 to 100 pg/ml, depending on the antibody system used. These concentrations (3–6 × 10−11 M) are of similar magnitude to thosereported to affect reputed target tissues of somatostatin in vitro and are thus consistent with a possible hormonal function for the peptide.

Glucose clearance (glucose utilization ÷ plasma glucose) is commonly used to assess glucose utilization under conditions in which plasma glucose concentrations vary. The validity of this practice requires that glucose clearance itself be independent of plasma glucose concentration. The present studies were, therefore, undertaken to determine the relationship between glucose clearance and plasma glucose concentration in man. Using the glucose clamp technique, rates of glucose utilization (measured isotopically with 3-3H-glucose) and glucose clearance were determined in 5 normal volunteers at steady-state plasma glucose concentrations of approximately 60, 95,130, and 165 mg/dl, while plasma insulin concentrations were maintained constant (∼18 μU/ml) by infusion of insulin and somatostatin. Despite virtually identical 0.4 mg · kg−1 · min−1 increments in glucose utilization for each 35-mg/dl increment in plasma glucose, glucose clearance decreased as a function of plasma glucose concentration (r = − 0.85, P < 0.001). These results indicate that glucose clearance is not independent of changes in plasma glucose concentration and, thus, use of glucose clearance to evaluate glucose utilization at differing plasma glucose concentration is not valid. Whether this conclusion also applies to similar use of clearance for other substrates remains to be determined.

To determine the mechanism by which hyperinsulinemia causes hypoglycemia in insulinoma patients, rates of glucose production and utilization, and circulating levels of insulin, glucagon, alanine, lactate, and glycerol were measured in 6 insulinoma patients during development of fasting hypoglycemia and in 8 normal volunteers studied over an identical interval. Initially, insulinoma patients had a greater plasma insulin (42 ± 9 versus 15 ± 1 μU/ml) and glucagon levels (214 ± 31 versus 158 ± 21 pg/ml) than normal subjects, P < 0.05, but their plasma glucose levels (81 ± 4 mg/dl) and rates of glucose production and utilization (1.71 ± 0.08 and 1.74 ± 0.08 mg/kg min, respectively) were not significantly different from those of normal subjects (93 ± 2 mg/dl, 1.93 / 0.11, and 1.92 ± 0.13 mg/kg · min, respectively). During a subsequent 8-h fast, glucose production and glucose utilization decreased in both groups, but more markedly in insulinoma patients. Since glucose utilization exceeded glucose production to a greater extent in insulinoma patients than in normal subjects, plasma glucose decreased to 44 ± 3 mg/dl in insulinoma patients, but only to 84 ± 1 mg/dl in normal subjects (P < 0.001). Glucose utilization in insulinoma patients never exceeded that of normal subjects. These results demonstrate that fasting hypoglycemia in the insulinoma patients is usually due to suppression of glucose production rather than to acceleration of glucose utilization, as is widely thought. A direct effect of insulin on the liver is probably responsible, since circulating levels of gluconeogenic precursors are normal and since plasma glucagon increases during development of hypoglycemia in insulinoma patients.

A new bioartificial pancreas, in which rat islets within a Millipore chamber are perifused with blood ultrafiltrate (polyacrylonitrile membrane), has been developed and its function evaluated in vitro and in vivo. Use of ultrafiltration (instead of diffusion) permitted rapid transmission of changes in blood glucose to the islet compartment (less than 10 min). To determine whether ultrafiltrate of Krebs-Ringer bicarbonate buffer (KRBB) and blood could support normal islet Bcell function, insulin secretion of freshly isolated islets in response to a 20-mM glucose challenge was examined during perifusion with KRBB and then with ultrafiltrate produced either from KRBB itself or from blood of normal and streptozotocin-induced diabetic rats. In the latter case, perifusate was reinfused to the animals, whose plasma glucose and insulin were then measured. Insulin responses to glucose were virtually identical when the same islets were perifused with KRBB and then with ultrafiltrate of KRBB (47 ± 12 versus 41 ± 9 ng/100 islets/24 min), or when islets were perifused with KRBB and then with ultrafiltrate produced from blood of normal rats (69 ± 6 versus 73 ± 11 ng/100 islets/24 min). When five diabetic rats were connected to the device containing 600 islets, their plasma glucose decreased from 19.8 ± 1.2 to 6.1 ± 0.5 mM (P < 0.001) within 120 min without overshoot hypoglycemia, and remained normal for up to 6 h. Plasma insulin increased from 2.0 ± 0.1 to 11.4 ± 1.4 ng/ml (P < 0.02) at 30 min and then decreased to 2.9 ± 0.6 ng/ml. After intravenous glucose administration (0.5 g/kg, N = 4), plasma insulin increased twofold within 15 min, and glucose disappearance rates were identical to those obtained in four normal rats (K = 2.13 ± 0.18 versus 2.23 ± 0.09, NS). These results indicate that ultrafiltrate of KRBB and blood can acutely support normal islet B-cell function, and that ultrafiltration can provide satisfactory glucose-insulin kinetics for an implantable closed-loop insulin delivery system, in which nonsyngeneic islets may be protected against immune rejection.

To determine whether receptor and/or postreceptor abnormalities of insulin action were responsible for insulin resistance in nonobese patients with non-insulindependent diabetes mellitus (NIDDM) and to assess the role of insulin resistance in their impaired glucose tolerance, insulin dose-response characteristics, insulin binding to monocytes, and insulin secretion were compared in 10 nonobese patients with NIDDM and six age-weight-matched nondiabetic volunteers. The insulin resistance of the diabetics was characterized by a shift to the right of their insulin dose-response curve (Km 81 ± 4 μU/ml vs. 58 ± 2 μU/ml in the nondiabetics P < 0.001) but a normal maximal response to insulin. Although monocyte insulin binding was decreased in the diabetics (P < 0.01), their response to insulin was appropriate for the number of insulin receptors occupied indicating normal postreceptor function. Insulin secretion was markedly reduced in diabetic subjects (52 ± 22 vs. 471 ± 90μu.U · ml−1 · 10 mirr1 in the nondiabetic subjects, P < 0.001) and was more strongly correlated with fasting plasma glucose (r = 0.92, P < 0.001) and intravenous glucose tolerance (Kivgtt) (r = 0.98, P < 0.001) than was insulin sensitivity (Km) (r = 0.23, NS, and r = 0.57, P < 0.05, respectively). We conclude that in nonobese patients with NIDDM, insulin resistance is characterized by a shift to the right of the insulin dose-response curve, which can be accounted for solely by an insulin receptor defect. However, in these patients, impaired insulin secretion rather than insulin resistance appears to be the predominant metabolic abnormality.