John W. Ensinck

Somatostatin: Hypothalamic Inhibitor of the Endocrine Pancreas

Somatostatin, a hypothalamic peptide that inhibits the secretion of pituitary growth hormone, inhibits basal insulin secretion in fasted cats and rats. In fasted baboons both basal and arginine-stimulated secretion of insulin and glucagon are inhibited. Somatostatin appears to act directly on the endocrine pancreas. The action is dose-related, rapid in onset, and readily reversed.

Evidence of Cosecretion of Islet Amyloid Polypeptide and Insulin by β-Cells

Islet amyloid polypeptide (IAPP) has been identified as the major constituent of the pancreatic amyloid of non-insulin-dependent diabetes mellitus (NIDDM) and is also present in normal β-cell secretory granules. To determine whether IAPP is a pancreatic secretory product, we measured the quantity of lAPP-like immunoreactivity (IAPP-LI), insulin, and glucagon released into 5 ml of incubation medium during a 2-h incubation of monolayer cultures (n = 5) of neonatal (3- to 5-day-old) Sprague-Dawley rat pancreases under three conditions: 1.67 mM glucose, 16.7 mM glucose, and 16.7 mM glucose plus 10 mM arginine and 0.1 mM isobutylmethylxanthine (IBMX). The quantity of IAPP-LI, insulin, and glucagon in the cell extract was also determined. Mean ± SE IAPP-LI in the incubation medium increased from 0.041 ± 0.003 pmol in 1.67 mM glucose to 0.168 ± 0.029 pmol in 16.7 mM glucose (P < 0.05) and 1.02 ± 0.06 pmol in 16.7 mM glucose plus arginine and IBMX (P < 0.05 vs. 1.67 or 16.7 mM glucose). Insulin secretion increased similarly from 4.34 ± 0.27 to 20.2 ± 0.6 pmol (P < 0.05) and then to 135 ± 5 pmol (P < 0.05 vs. 1.67 or 16.7 mM glucose). Glucagon release tended to decrease with the increase in glucose concentration (0.39 ± 0.01 vs. 0.33 ± 0.02 pmol, P < 0.1), whereas with the addition of arginine and IBMX to high glucose, glucagon release increased to 1.32 ± 0.03 pmol (P < 0.05 vs. 1.67 or 16.7 mM glucose). Thus, the molar proportion of IAPP-LI to insulin secreted in low glucose was ∼1% and did not differ significantly with stimulation (0.95 ± 0.08 vs. 0.84 ± 0.15 vs. 0.76 ± 0.05%). In contrast, there was no constant proportional relationship between the release of IAPP-LI and glucagon (10.6 ± 0.8 vs. 51.3 ± 8.7 vs. 77.5 ± 5.2%). After incubation in 1.67 mM glucose, the extracted cells contained 3.7 ± 0.2 pmol IAPP-LI, 944 ± 25 pmol insulin, and 28.2 ±1.5 pmol glucagon. After maximal stimulation, the fractional release of IAPP-LI was 26.7 ± 0.7% vs. 14.7 ± 0.6% of insulin and 4.4 ± 0.2% of glucagon. These data indicate that nondiabetic neonatal rat islet cultures contain IAPP-LI and release it after stimulation by glucose and nonglucose secretagogues. Furthermore, the data suggest that IAPP-LI is a product of the β-cell, which coreleases it with insulin in a molar ratio of ∼1.100.

Glucagon-like peptide 1 enhances glucose tolerance both by stimulation of insulin release and by increasing insulin-independent glucose disposal.

Glucagon-like peptide 1 [7-36 amide] (GLP-1) has been shown to enhance insulin secretion in healthy and type II diabetic humans, and to increase glucose disposal in type I diabetic patients. To further define its action on glucose kinetics, we studied six healthy subjects who received either GLP-1 (45 pmol/kg per h) or 150 mM saline on two mornings during which a modified intravenous glucose tolerance test was performed. Plasma insulin and glucose levels were analyzed using Bergmans minimal model of glucose kinetics to derive indices of insulin sensitivity (SI) and glucose effectiveness at basal insulin (SG), the latter a measure of glucose disposition independent of changes in insulin. In addition, basal insulin concentrations, the acute insulin response to glucose (AIRg), plasma glucagon levels, and the glucose disappearance constant (Kg) were measured on the days that subjects received GLP-1 or saline. Compared with saline infusions, GLP-1 increased the mean Kg from 1.61 +/- 0.20 to 2.65 +/- 0.25%/min (P = 0.022). The enhanced glucose disappearance seen with GLP-1 was in part the result of its insulinotropic effect, as indicated by a rise in AIRg from 240 +/- 48 to 400 +/- 78 pM (P = 0.013). However, there was also an increase in SG from 1.77 +/- 0.11 to 2.65 +/- 0.33 x 10(-2).min-1 (P = 0.038), which was accounted for primarily by insulin-independent processes, viz glucose effectiveness in the absence of insulin. There was no significant effect of GLP-1 on SI or basal insulin, and glucagon levels were not different during the glucose tolerance tests with or without GLP-1. Thus, GLP-1 improves glucose tolerance both through its insulinotropic action and by increasing glucose effectiveness. These findings suggest that GLP-1 has direct effects on tissues involved in glucose disposition. Furthermore, this peptide may be useful for studying the process of insulin-independent glucose disposal, and pharmacologic analogues may be beneficial for treating patients with diabetes mellitus.

Autonomic Neural Dysfunction in Recently Diagnosed Diabetic Subjects

Because onset of autonomic neural dysfunction in the diabetic syndrome has not been well established, sensitive and quantitative measures of autonomic nervous system (ANS) function were made in 19 non-insulin-dependent (NIDD) and 14 insulin-dependent (IDD) recent-onset diabetic subjects. The known duration of diabetes mellitus in the NIDD subjects was ≤ 12 mo. The duration in the IDD subjects was ≤ 24 mo. RR-variation during beta adrenergic blockade (an index of an ANS reflex involving the cardiac parasympathetic nervous system [PNS] pathway) was smaller than that of control subjects in both NIDD (P < 0.001) and IDD subjects (P < 0.01). This PNS abnormality was not likely to be due to volume depletion since acute volume depletion induced by furosemide in six normal subjects (1608 ± 105 ml, mean ± SEM) did not change RR-variation. Dark-adapted pupil size after topical PNS blockade (an index of iris sympathetic nervous system [SNS] activity) was also smaller in both groups of diabetic subjects (NIDD, P < 0.01; IDD, P < 0.05). Pupillary latency time (an index of an ANS reflex involving iris PNS pathway) was prolonged in the NIDD subjects (P < 0.005) but was not significantly altered in the IDD subjects. Thus, it would appear that the ANS is impaired soon after the diagnosis of diabetes mellitus. We hypothesize that early impairment of the ANS is common in IDD and NIDD subjects. This finding is consistent with the hypothesis that abnormal carbohydrate metabolism is an important factor in the etiology of diabetic autonomic neuropathy.

Arginine-stimulated acute phase of insulin and glucagon secretion in diabetic subjects.

To determine if both phases of glucagon secretion are excessive in diabetes, arginine was admimistered intravenously as pulses and as infusions to normal subjects, insulin-dependent diabetics, and noninsulin-requiring diabetics. The acute phase of glucagon secretion, in response to arginine pulses at four different doses (submaximal to maximal alpha-cell stimulating), was indistinguishable in terms of timing, peak levels attained, and total increments comparing controls and diabetics. During the first half of the arginine infusion (500 mg/kg over 30 min) the glucagon rise in controls and diabetics was similar (P greater than 0.1), whereas during the last half of the infusion excessive glucagon levels were seen in the diabetics. No difference in the glucagon responses to arginine administered as either a pulse or an infusion was observed between the two types of diabetics. The acute phase responses of insulin to intravenous, maximal stimulating doses of glucose (20 g) and arginine (2.5 g) were measured in five insulin-independent diabetics. Although the acute insulin response to arginine was normal, there was marked attentuation of the early beta-cell response upon stimulation by glucose. From these results we conclude that although in diabetes excessive glucagon levels are observed with chronic arginine stimulation, the acute phase of glucagon secretion in response to arginine is normal. In addition, the beta-cell in noninsulin-requiring diabetics, although acutely hyporesponsive to glucose, remains normally responsive to another stimulus, arginine.

Elimination of the action of glucagon-like peptide 1 causes an impairment of glucose tolerance after nutrient ingestion by healthy baboons.

Glucagon-like peptide 1 (GLP-1) is an insulinotropic hormone released after nutrient ingestion which is known to augment insulin secretion, inhibit glucagon release, and promote insulin-independent glucose disposition. To determine the overall effect of GLP-1 on glucose disposition after a meal we studied a group of healthy, conscious baboons before and after intragastric glucose administration during infusions of saline, and two treatments to eliminate the action of GLP-1: (a) exendin-[9-39] (Ex-9), a peptide receptor antagonist of GLP-1; or (b) an anti-GLP-1 mAb. Fasting concentrations of glucose were higher during infusion of Ex-9 than during saline (4.44 +/- 0.05 vs. 4.16 +/- 0.05 mM, P < 0.01), coincident with an elevation in the levels of circulating glucagon (96 +/- 10 vs. 59 +/- 3 ng/liter, P < 0.02). The postprandial glycemic excursions during administration of Ex-9 and mAb were greater than during the control studies (Ex-9 13.7 +/- 2.0 vs. saline 10.0 +/- 0.8 mM, P = 0.07; and mAb 13.6 +/- 1.2 vs. saline 10.6 +/- 0.9 mM, P = 0.044). The increments in insulin levels throughout the absorption of the glucose meal were not different for the experimental and control conditions, but the insulin response in the first 30 min after the glucose meal was diminished significantly during treatment with Ex-9 (Ex-9 761 +/- 139 vs. saline 1,089 +/- 166 pM, P = 0.044) and was delayed in three of the four animals given the neutralizing antibody (mAb 946 +/- 262 vs. saline 1,146 +/- 340 pM). Thus, elimination of the action of GLP-1 impaired the disposition of an intragastric glucose meal and this was at least partly attributable to diminished early insulin release. In addition to these postprandial effects, the concurrent elevation in fasting glucose and glucagon during GLP-1 antagonism suggests that GLP-1 may have a tonic inhibitory effect on glucagon output. These findings demonstrate the important role of GLP-1 in the assimilation of glucose absorbed from the gut.

Enteral Enhancement of Glucose Disposition by Both Insulin-Dependent and Insulin-Independent Processes: A Physiological Role of Glucagon-Like Peptide I

Glucagon-like peptide I (GLP-I)(7–36) amide is secreted by intestinal L-cells in response to food ingestion. GLP-I is a potent insulin secretagogue and also inhibits glucagon release. Inaddition, when given to humans in pharmacological amounts, GLP-I increases glucose disposal independent of its effects on islet hormone secretion. To test the hypothesis that this extrapancreatic effect of GLP-I on glucose disposition is present at physiological levels of GLP-I, we performed intravenous glucose tolerance tests (IVGTTs) 1 h after the following interventions: 1) the ingestion of 50 g fat to stimulate GLP-I secretion or the ingestion of water as a control and 2) infusion of GLP-I to attain physiological levels or a control infusion of saline. The results of the IVGTTs were analyzed using the minimal model technique to determine the insulin sensitivity index (SI) and indexes of insulin-independent glucose disposition, glucose effectiveness at basal insulin (SG), and glucose effectiveness at zero insulin (GEZI), as wellas the glucose disappearance constant (kg) and the acute insulin response to glucose (AIRg). These parameters were compared between conditions of elevated circulating GLP-I and control conditions. After ingestion of fat and infusion of synthetic hormone, plasma GLP-I increased to similar levels; GLP-I did not change with water ingestion or saline infusion. Elevated levels of GLP-I, whether from fat ingestion or exogenous infusion, caused increased glucose disappearance (kg: fat versus water 2.67 ± 0.2 vs. 1.72 ± 0.2, P < 0.001; GLP-I versus saline 2.42 ± 0.2 vs. 1.96 ± 0.2 %/min, P = 0.045), insulin secretion (AIRg: fat versus water 427 ± 50 vs. 284 ± 41, P = 0.001; GLP-I versus saline 376 ± 65 vs. 258 ± 16 pmol/1, P = 0.03), and glucose effectiveness (SG: fat versus water 2.5 ± 0.1 vs. 1.8 ± 0.2, P = 0.001; GLP-I versus saline 2.5 ± 0.2 vs. 1.8 ± 0.2%/min, P = 0.014; GEZI: fat versus water 1.9 ± 0.2 vs. 1.3 ± 0.2%/min, P = 0.003; GLP-I versus saline 1.9 ± 0.2 vs. 1.3 ± 0.2,P = 0.006) but no difference in insulin sensitivity. These results suggestthat GLP-I, released after meals, promotes glucose assimilation both by augmenting insulin secretion and through a separate effect to increase glucose uptake and/or inhibit hepatic glucose output.

Arginine-stimulated Acute Phase of Insulin and Glucagon Secretion: I. In Normal Man

To document and characterize the immediate phase of glucagon secretion as detected in peripheral blood in man, we have given pulses of L-arginine (0.1 gm. to 10.0 gm.) intravenously over twenty to thirty seconds to twenty-three healthy young men. Peak glucagon and insulin levels averaging four and five times basal levels respectively were reached two to five minutes after arginine administration and had returned to baseline levels by fifteen to thirty minutes. Computing the area above basal for the initial ten minutes after arginine stimulation established a dose-response relationship for the acute phases of glucagon and insulin secretion. A maximal glucagon response was elicited by doses of arginine of 5.0 gm. or greater, whereas for insulin, the plateau was reached at 2.5 gm. of arginine. Sequential 5.0-gm. pulses of arginine administered every thirty minutes showed that there was no augmentation or attenuation of the timing, magnitude (area 0-10 minutes) or absolute peak values reached for either the glucagon or insulin responses. The effect of induced hyperglycemia on the acute phase of insulin and glucagon secretion was assessed by administering the arginine during marked elevation of ambient glucose concentration achieved by the intravenous administration of glucose. This resulted in marked suppression of the acute glucagon response and dramatic accentuation of the insulin response.

Elevated Plasma Glucose and Lowered Triglyceride Levels From Omega-3 Fatty Acid Supplementation in Type II Diabetes

We studied the effect of omega-3 fatty acids (ω3FA) on glucose homeostasis and lipoprotein levels in eight type II (non-insulin-dependent) diabetic subjects ingesting 8 g/day to3FA for 8 wk as marine-lipid concentrate capsules. After ω3FA supplementation, fasting plasma glucose levels increased 22% (P = .005) and meal-stimulated glucose increased 35% (P = .036). The percentage of glucose elevation correlated with percentage ideal body weight (r = .73, P = .04). No significant changes were seen in fasting or meal-stimulated plasma insulin, glucose disposal, or insulin-to-glucagon ratios. Very-low-density lipoprotein cholesterol and triglyceride (TG) levels showed consistent reductions of 56% (P < .001) and 42% (P < .001), respectively, after ω3FA supplementation. Total cholesterol levels decreased 7% (P < .05) without alteration in low- or high-density lipoprotein cholesterol. Thus, ω3FA supplementation at a dose of 8 g/day significantly improves plasma TG levels but increases fasting and meal-stimulated glucose concentrations in the type II diabetic patient not treated with insulin or sulfonylurea agents. Marine-lipid concentrate capsules supplying large amounts of ω3FAs should be used cautiously in the type II diabetic patient.

The Effect of Adrenergic Blockade on the Glucagon Responses to Starvation and Hypoglycemia in Man

In an attempt to ascertain whether the sympathetic nervous system modulates glucagon release in man during starvation and hypoglycemia, the influence of alpha and beta adrenergic blockade on glucagon responses was studied in young, healthy men subjected to fasting and insulin-induced hypoglycemia. Six volunteers fasted for 84 h on three separate occasions. Plasma immunoreactive glucagon (IRG), measured initially at 12 h, climbed gradually from mean levels of 54 pg/ml to a zenith of 124 pg/ml at 48 h, with maintenance of these levels for the duration of the fast. The infusion of propranolol or phentolamine throughout the terminal 24 h of the second and third fasts failed to alter the pattern of IRG release. After an overnight fast, five volunteers received insulin intravenously, which evoked a mean rise in plasma IRG levels from 63 pg/ml to a maximum of 256 pg/ml at 30 min. The concurrent administration of propranolol or phentolamine did not modify the glucagon responses to insulin-induced hypoglycemia. These data suggest that the augmented glucagon release in man during starvation or after hypoglycemia is not significantly regulated by signals from the adrenergic nervous system.