Citations

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.

In vitro perfusion and incubation studies and recent investigations in dogs suggest that branched chain amino acids (BCAA) may be a major source of alanine nitrogen. To determine the contribution of BCAA nitrogen to the formation of alanine in man, seven postabsorptive adults received prime-dose constant infusions of 15N-leucine, L-[6,6,6-2H3] leucine, and L-[2,3,3,3-2H4] alanine; isotopic enrichment was determined in arterialized venous plasma samples by gas chromatography-mass spectroscopy. At substrate and isotope steady state, alanine flux and the rate of 15N alanine appearance were 5.4 ± 0.3 μmol/kg-min and 32 ± 2 nmol/kg·min, respectively. Leucine nitrogen flux was significantly > that of leucine carbon flux (2.54 ± 0.25 vs. 1.90 ± 0.10 μmol/kg·min, respectively; P < 0.001). The 30% greater flux of leucine nitrogen when compared with leucine carbon suggests significant recycling of the leucine carbon in vivo. The percent of circulating alanine nitrogen derived from leucine was 12.5 ± 1.5%; however, the rate of leucine nitrogen transferred to alanine was 0.66 ± 0.05 mumol/kg.min, and represents a minimum of 28% of leucine nitrogen going to alanine. On the basis of these data, together with the percent of alanine and leucine in body protein, only 40% of circulating plasma alanine could come from endogenous protein, whereas 60% is derived from de novo synthesis. In addition, at least 20% of the nitrogen required for alanine synthesis is derived solely from leucine following an overnight fast. Therefore, if the contribution of isoleucine and valine nitrogen is similar to that of leucine, the BCAA may contribute to a minimum of 60% of the nitrogen required for alanine synthesis in postabsorptive man.

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.

Rapid axonal transport was studied by several methods in rats with serum glucose levels above 300 mg/dl as a result of treatment with streptozotocin (45–50 mg/kg) 3 days, 1 wk, 4 wk, 8 wk, or 4 mo earlier. With untreated age-matched rats as controls, rapid anterograde axonal transport in the sciatic nerves of diabetic rats appeared entirely normal. Statistically significant differences were never observed between experimental and control nerves in the basal content of acetylcholinesterase (AChE) and dopamine-β-hydroxylase (DBH) activity or in the rate of accumulation of these enzymes proximal or distal to a ligature. Therefore, the basic capacity for rapid anterograde and retrograde axonal transport of proteins was probably unimpaired in the diabetic nerves. The accumulation of labeled glycoproteins proximal to ligatures on the contralateral nerves of the same rats after injection of the L5 dorsal root ganglion with 3H-fucose was likewise normal. However, rats with 1, 4, and 8 wk of diabetes did show reduced accumulation of fucose-labeled protein distal to nerve ligations, indicating a long-lasting abnormality of retrograde axonal transport. Furthermore, this abnormality was reversed by daily insulin treatment during the second half of an 8-wk experimental period. It is therefore unlikely that the depression of retrograde transport reflects direct toxic effects of streptozotocin. We conclude that streptozotocin-induced diabetes leads to: (1) abnormal delay in the turnaround of transported proteins in distal nerve regions, perhaps combined with (2) an abnormal metabolism of glycoproteins. A delayed onset of retrograde transport is consistent with present observations of reduced accumulation of 125I-labeled nerve growth factor (NGF) below a midthigh ligature on the sciatic nerve after injection of this protein into the hindfoot of rats with 3–5 wk of diabetes. Further work on the factors controlling rapid retrograde transport of proteins in diabetic nerve is warranted.

To assess the effects of endogenous somatostatin on pancreatic islet A- and B-cell function, isolated rat islets were incubated in antisomatostatin γ-globulin to bind endogenously released somatostatin, and the insulin and glucagon secretion of these islets was compared with that of islets incubated in γ-globulin isolated from nonimmune serum. Islets incubated in antisomatostatin γ-globulin released significantly more insulin at 4, 8,16, and 32 mM glucose and significantly more glucagon at 8,16, and 32 mM glucose, P < 0.05–0.005. For glucose-stimulated insulin release the threshold was decreased, the Vmax was increased, but the apparent Km was unaltered; for glucose-suppression of glucagon release the threshold was increased, maximal suppression was decreased, but the apparent Ki was unaltered. The augmentative effect of the antisomatostatin γ-globulin was most prominent at 4 mM glucose for insulin release and at 8 mM glucose for glucagon release, but was not limited to glucose since both insulin and glucagon release stimulated by arginine were also augmented by antisomatostatin γ-globulin. These results provide evidence that endogenous somatostatin may act as a physiologic local regulator of both insulin and glucagon secretion and that its effect on insulin and glucagon secretion is dependent on the prevailing glucose concentration.