J. A. Galloway

Abnormal Patterns of Insulin Secretion in Non-Insulin-Dependent Diabetes Mellitus

To determine whether non-insulin-dependent diabetes is associated with specific alterations in the pattern of insulin secretion, we studied 16 patients with untreated diabetes and 14 matched controls. The rates of insulin secretion were calculated from measurements of peripheral C-peptide in blood samples taken at 15- to 20-minute intervals during a 24-hour period in which the subjects ate three mixed meals. Incremental responses of insulin secretion to meals were significantly lower in the diabetic patients (P less than 0.005), and the increases and decreases in insulin secretion after meals were more sluggish. These disruptions in secretory response were more marked after dinner than after breakfast, and a clear secretory response to dinner often could not be identified. Both the control and diabetic subjects secreted insulin in a series of discrete pulses. In the controls, a total of seven to eight pulses were identified in the period from 9 a.m. to 11 p.m., including the three post-meal periods (an average frequency of one pulse per 105 to 120 minutes), and two to four pulses were identified in the remaining 10 hours. The number of pulses in the patients and controls did not differ significantly. However, in the patients, the pulses after meals had a smaller amplitude (P less than 0.03) and were less frequently concomitant with a glucose pulse (54.7 +/- 4.9 vs. 82.2 +/- 5.0, P less than 0.001). Pulses also appeared less regularly in the patients. During glucose clamping to produce hyperglycemia (glucose level, 16.7 mmol per liter [300 mg per deciliter]), the diabetic subjects secreted, on the average, 70 percent less insulin than matched controls (P less than 0.001). These data suggest that profound alterations in the amount and temporal organization of stimulated insulin secretion may be important in the pathophysiology of beta-cell dysfunction in diabetes.

Quantitative study of insulin secretion and clearance in normal and obese subjects.

The secretion and hepatic extraction of insulin were compared in 14 normal volunteers and 15 obese subjects using a previously validated mathematical model of insulin secretion and rate constants for C-peptide derived from analysis of individual decay curves after intravenous bolus injections of biosynthetic human C-peptide. Insulin secretion rates were substantially higher than normal in the obese subjects after an overnight fast (86.7 +/- 7.1 vs. 50.9 +/- 4.8 pmol/m2 per min, P less than 0.001, mean +/- SEM), over a 24-h period on a mixed diet (279.6 +/- 24.2 vs. 145.8 +/- 8.8 nmol/m2 per 24 h, P less than 0.001), and during a hyperglycemic intravenous glucose infusion (102.2 +/- 10.8 vs. 57.2 +/- 2.8 nmol/m2 per 180 min, P less than 0.001). Linear regression analysis revealed a highly significant relationship between insulin secretion and body mass index. Basal hepatic insulin extraction was not significantly different in the normal and obese subjects (53.1 +/- 3.8 vs. 51.6 +/- 4.0%). In the normal subjects, fasting insulin did not correlate with basal hepatic insulin extraction, but a significant negative correlation between fasting insulin and hepatic insulin extraction was seen in obesity (r = -0.63, P less than 0.02). This finding reflected a higher extraction in the six obese subjects with fasting insulin levels within the range of the normal subjects than in the nine subjects with elevated fasting insulin concentrations (61 +/- 3 vs. 45 +/- 6%, P less than 0.05). During the hyperglycemic clamp, the insulin secretion rate increased to an average maximum of 6.2-fold over baseline in the normal subjects and 5.8-fold in the obese subjects. Over the same time, the peripheral insulin concentration increased 14.1-fold over baseline in the normals and 16.6-fold over baseline in the obese, indicating a reduction in the clearance of endogenously secreted insulin. Although the fall in insulin clearance tended to be greater in the obese subjects, the differences between the two groups were not statistically significant. Thus, under basal, fasting conditions and during ingestion of a mixed diet, the hyperinsulinemia of obesity results predominantly from increased insulin secretion. In patients with more marked basal hyperinsulinemia and during intense stimulation of insulin secretion, a reduction in insulin clearance may contribute to the greater increase in peripheral insulin concentrations that are characteristic of the obese state.+

Use of biosynthetic human C-peptide in the measurement of insulin secretion rates in normal volunteers and type I diabetic patients.

We undertook this study to examine the accuracy of plasma C-peptide as a marker of insulin secretion. The peripheral kinetics of biosynthetic human C-peptide (BHCP) were studied in 10 normal volunteers and 7 insulin-dependent diabetic patients. Each subject received intravenous bolus injections of BHCP as well as constant and variable rate infusions. After intravenous bolus injections the metabolic clearance rate of BHCP (3.8 +/- 0.1 ml/kg per min, mean +/- SEM) was not significantly different from the value obtained during its constant intravenous infusion (3.9 +/- 0.1 ml/kg per min). The metabolic clearance rate of C-peptide measured during steady state intravenous infusions was constant over a wide concentration range. During experiments in which BHCP was infused at a variable rate, the peripheral concentration of C-peptide did not change in proportion to the infusion rate. Thus, the infusion rate of BHCP could not be calculated accurately as the product of the C-peptide concentration and metabolic clearance rate. However, the non-steady infusion rate of BHCP could be accurately calculated from peripheral C-peptide concentrations using a two-compartment mathematical model when model parameters were derived from the C-peptide decay curve in each subject. Application of this model to predict constant infusions of C-peptide from peripheral C-peptide concentrations resulted in model generated estimates of the C-peptide infusion rate that were 101.5 +/- 3.4% and 100.4 +/- 2.8% of low and high dose rates, respectively. Estimates of the total quantity of C-peptide infused at a variable rate over 240 min based on the two-compartment model represented 104.6 +/- 2.4% of the amount actually infused. Application of this approach to clinical studies will allow the secretion rate of insulin to be estimated with considerable accuracy. The insulin secretion rate in normal subjects after an overnight fast was 89.1 pmol/min, which corresponds with a basal 24-h secretion of 18.6 U.

The effects of biosynthetic human proinsulin on carbohydrate metabolism.

Large quantities of biosynthetic human proinsulin have recently become available through recombinant DNA technology. Since the in vivo effects of human proinsulin have not been studied in man, we compared the dose-response relationship for stimulation of glucose disposal and suppression of hepatic glucose output by proinsulin and insulin. Ten normal subjects were studied using the euglycemic glucose clamp technique. The human proinsulin and insulin infusion rates were chosen to achieve steady-state proinsulin levels 10-fold higher than insulin levels on a molar basis, based on previous observations that porcine proinsulin has ∼10% the potency of insulin. Proinsulin infusion rates of 2.75, 7.5, 22.5, and 45 μg/m2/min were compared with insulin infusion rates of 0.63,1.67, 5, and 10 μg/ m2/min. Primed, continuous infusions of insulin yielded steady-state levels within 25 min, whereas proinsulin levels did not reach a steady state for 120–180 min. The metabolic clearance rate of insulin was 11–12 ml/kg/min at the lower infusion rates but fell to 8.4 ml/kg/min at the highest infusion rate. The metabolic clearance rate of proinsulin was 3.0–3.5 ml/kg/ min at all infusion rates. Dose-response analysis demonstrated that proinsulin-mediated glucose disposal was ∼8% that of insulin. In contrast, proinsulin-mediated suppression of hepatic glucose output was ∼12% that seen with insulin. We conclude that: (1) primed, continuous infusions of proinsulin and insulin have considerably different kinetics; (2) the metabolic clearance rate of proinsulin is only −28% that of insulin, but the processes responsible for removal of insulin from the circulation are more saturable than those responsible for removal of proinsulin; and (3) proinsulin appears to exert a greater effect on hepatic than on peripheral tissues.

Insulin Secretion and Clearance: Comparison After Oral and Intravenous Glucose

Insulin secretion and clearance in response to the administration of oral and intravenous glucose was investigated in nine normal men. C-peptide metabolic kinetics were calculated by analysis of individual C-peptide decay curves after the bolus injection of biosynthetic human C-peptide. Glucose was administered to the subjects on three occasions: as a 75-g oral dose, a 75-g i.v. infusion, and an intravenous glucose infusion at a variable rate adjusted to mimic the peripheral glucose levels obtained after the oral glucose load (matching experiment). Glucose, insulin, and C-peptide concentrations were measured for the subsequent 5 h. The glucose level after the oral glucose load (115.9 ± 2.6 mg/dl, mean ± SE) closely approximated that after the matching experiment (120.5 ± 2.5 mg/dl) but was significantly lower than after 75 g i.v. glucose (127.7 ± 3.4 mg/dl, P < .05). Analysis of the areas under the peripheral concentration curves (60-360 min) showed that the responses of both insulin (52.7 ± 5.6 and 46.5 ± 4.5 pmol · ml−1 300 min1) and C-peptide (252.7 ± 27.5 and 267.0 ± 21.6 pmol · ml−1 · 300 min1) were not significantly different after the oral and 75-g i.v. glucose studies, respectively, whereas in the matching experiment, both the insulin (26.1 ± 3.9 pmol · ml−1 · 300 min−1) and C-peptide (178.0 ± 18.9 pmol ml−1 300 min−1) responses were lower (P < .05) than in the other two studies. Insulin secretory rates were derived from peripheral C-peptide concentrations with an open two-compartment model and individually derived model parameters. The basal insulin secretion rate was 86.8 ± 2.9 pmol/min. The insulin secretory response over the 300 min was 66.2 ± 4.8 nmol after oral glucose. This was similar to that after 75 g i.v. glucose (72.4 ± 4 . 1 nmol), whereas that secreted in response to the matching experiment was lower (47.6 ± 4.1 nmol, P ± .05). As a measure of the clearance of endogenous insulin, the ratio between the area under the insulin secretory curve and the area under the peripheral insulin concentration curve was calculated. This ratio was similar (1906 ± 149 ml/min) during the baseline period and the matching glucose infusion (2042 ± 245 ml/min) but was significantly lower after oral glucose (1330 ±112 ml/min, P < .05). The incretin effect calculated based on the insulin secretion rate (25 ± 9.2%) appeared to be less than if the calculations were based on peripheral insulin levels. These data demonstrate that equivalent doses of glucose administered orally and intravenously elicit an equivalent insulin secretory response. However, when the arterialized plasma glucose curve after 75 g oral glucose is matched by an intravenous glucose infusion, only 35.6 ± 2.9 g glucose was infused, and the intravenous glucose resulted in a lower secretory response. Furthermore, after oral administration of 75 g glucose a significant reduction in insulin clearance resulted. These data provide evidence that the hyperinsulinemia seen after oral glucose is due both to enhanced insulin secretion and diminished insulin clearance.

The Effects of Biosynthetic Human Proinsulin on Carbohydrate Metabolism in Non-Insulin-Dependent Diabetes Mellitus

We compared the glucose-lowering effect of proinsulin, the precursor molecule of insulin, with that of insulin itself. In patients with non-insulin-dependent diabetes mellitus (NIDDM) in whom proinsulin (0.2 U per kilogram of body weight) was subcutaneously injected at 9 a.m., fasting glucose levels (247 +/- 22 mg per deciliter [+/- SEM]) became normal within six hours and elevated rates of hepatic glucose output were lowered. The response to regular insulin (0.2 U per kilogram) was of similar magnitude but faster. Glucose clearance was stimulated less by proinsulin, reflecting its preferential action in suppressing glucose output. Hypoglycemia occurred in five of nine insulin-treated patients, but in only one of nine proinsulin-treated patients. After proinsulin injection at bedtime (30.5 +/- 4 U), serum proinsulin concentrations reached a peak by five hours and declined gradually thereafter. Fasting hepatic glucose output became normal, and euglycemia was sustained without overnight hypoglycemia. Proinsulin reduced plasma glucose more effectively than an equal unit dosage of NPH insulin, but since higher doses of NPH insulin were not used, no conclusions could be drawn about the relative desirability of these preparations for clinical use. We conclude that subcutaneously injected proinsulin has prolonged pharmacokinetics in plasma and can normalize plasma glucose in NIDDM characterized by severe hyperglycemia; as compared with the hypoglycemic effects of regular insulin, those of proinsulin are mostly due to suppression of hepatic glucose output, with little stimulation of glucose disposal and less hypoglycemia; and proinsulin may have a role in the treatment of NIDDM.

In Vivo Deactivation of Proinsulin Action on Glucose Disposal and Hepatic Glucose Production in Normal Man

We have studied the deactivation of the in vivo actions of insulin and biosynthetic human proinsulin (recombinant DNA) to stimulate the glucose disposal rate (GDR) and to inhibit hepatic glucose output (HGO) in man. Twelve healthy, lean, young subjects were studied using a modification of the euglycemic glucose clamp technique. Subjects received 4-h infusions on separate occasions of insulin (15 mU/m2/min equivalent to 0.54 μg/m2/min) or proinsulin (2.75 μg/m2/min), achieving steady-state serum levels of 32 ± 3 μU/ml (equivalent to 0.23 ± 0.02 pmol/ml) and 3.7 ± 0.2 pmol/ml, respectively. Suppression of HGO was similar (83–84%) with proinsulin and insulin, but stimulation of GDR above basal was greater with insulin (3.41 ± 0.43 versus 1.98 ± 0.28 mg/kg/min, P < 0.001). Following cessation of the hormone infusions, serum proinsulin concentration fell in a biphasic fashion with half-times of 25 and 146 min for the two phases. Serum half-disappearance time for insulin was 5 min. Deactivation of the hormones effects to stimulate GDR was 50% complete by 35 min after insulin and 71 min after proinsulin. In contrast, 50% of the recovery times for the effect on suppression of HGO were 55 min after insulin and 188 min after proinsulin. Serum glucagon levels did not differ significantly after the insulin and proinsulin infusions. In summary: (1) Deactivation of proinsulin and insulins effects to suppress HGO proceeds more slowly than deactivation of their effects to stimulate GDR; and (2) There is a markedly prolonged and disproportionately delayed deactivation of proinsulins effects on suppression of HGO. This later finding may prove of therapeutic value in the treatment of diabetes mellitus.

Reduction of Insulin Clearance During Hyperglycemic Clamp: Dose-Response Study in Normal Humans

Insulin secretion and clearance were studied in eight normal subjects who underwent hyperglycemie clamp studies at plasma glucose levels of 120, 225, and 300 mg/dl on three occasions. Insulin secretion rates were calculated during a 1-h baseline period and during 3 h of glucose clamping from a two-compartmental analysis of peripheral C-peptide concentrations with individual kinetic parameters derived after intravenous bolus injections of biosynthetic human C-peptide. At the 300-mg/dl clamp level, the insulin secretion rate increased to a value 9.9 ± 0.7 times that of basal at the end of the clamp (mean ± SE), whereas over the same period, the peripheral insulin concentrations increased to a greater extent, reaching a value 15.4 ± 1.2 times that of basal (P = .002). This greater relative increase in the insulin concentration in comparison with the corresponding insulin secretion rate suggests a reduction in the clearance of endogenous insulin. A similar trend was seen at the 225-mg/dl clamp level, but the relative increase in the insulin concentration (9.9 ±1.5 times that of basal) was not significantly higher than the relative increase in the insulin secretion rate (8.1 ± 0.5 times that of basal, P = .17). At the 120-mg/dl clamp level, the relative increases in the insulin secretion rate (2.7 ± 0.2 times that of basal) and the insulin concentration (2.4 ± 0.2 times that of basal) were similar (P = .26), indicating no reduction in endogenous insulin clearance during moderate stimulation of insulin secretion. In conclusion, a reduction in endogenous insulin clearance occurs during greater stimulation of insulin secretion at higher glucose-clamp levels. These data suggest that endogenous insulin clearance is nonlinear and shows evidence of saturation at high physiologic insulin concentrations.

Reevaluation of urine C-peptide as measure of insulin secretion.

Urine C-peptide (UCP) has been proposed as a measure of insulin secretion, because insulin and C-peptide are cosecreted in equimolar concentrations by the pancreatic β-cell. The validity of this approach was tested by comparing insulin secretion rates, calculated by application of a two-compartmental analysis of peripheral C-peptide concentrations, with UCP excretion rates. Insulin secretion and UCP excretion with subjects on a mixed diet were simultaneously measured over a 24-h period in 13 patients with non-insulin-dependent diabetes mellitus and in 14 matched nondiabetic control subjects. The fraction of secreted C-peptide that was excreted in the urine (fractional C-peptide excretion) showed considerable intersubject variability in the diabetic (11.3 ± 1.6%, range 3.9–20.8) and control (8.0 ± 1.7%, range 1.1–27.9, P = .07) subjects (means ± SE). UCP clearance demonstrated a similar degree of variability and was not significantly different (P = .07) between diabetic (23.8 ± 3.0 ml/min) and control (16.5 ± 2.7 ml/min) subjects. In control subjects, the 24-h insulin secretion rate correlated more closely with the fasting insulin secretion rate (r = .97, P = .0001), fasting C-peptide (r = .81, P = .0005), and fasting insulin (r = .80, P = .0005) concentrations than with the 24-h UCP excretion rate (r = .62, P = .02). Similar results were obtained in the diabetic patients. The mean coefficient of variation of fractional UCP excretion in 7 nondiabetic control subjects who were studied on a mixed diet over a 24-h period on two occasions was 28.4 ± 10.5%, that of UCP clearance was 28.9 ± 8.6%, and that of simultaneously measured creatinine clearance was 7.8 ± 3.5%. In summary, the fraction of secreted C-peptide that appears in the urine varies considerably between subjects and in the same subject studied repeatedly. UCP excretion does not correlate as well with 24-h insulin secretion as does the fasting insulin secretion rate or the fasting C-peptide or fasting insulin concentration. We conclude that, because the fraction of secreted C-peptide that is excreted in the urine varies considerably between subjects and in the same subject studied on different occasions, UCP is of only limited value as a quantitative measure of endogenous insulin secretion.

Lack of effect of high-dose biosynthetic human C-peptide on pancreatic hormone release in normal subjects

We studied the effect of high doses of biosynthetic human C-peptide on pancreatic hormone secretion in response to oral (75 g) and intravenous [( IV] 0.33 g/kg of D50%) glucose on normal volunteers. The infusion of human C-peptide at a rate of 360 ng/kg/min body weight, increased the plasma C-peptide concentration from a basal level of 0.32 +/- 0.04 pmol/mL to 38.5 +/- 1.8 pmol/ml. Overall, C-peptide had no significant effect on the serum levels of glucose, insulin, proinsulin, glucagon, and pancreatic polypeptide, either under basal conditions or following IV and oral glucose administration. However, small decreases in glucose and insulin concentrations that were not statistically significant were seen during the first hour after C-peptide infusion. The results of the present studies are therefore consistent with the conclusion that even supraphysiologic plasma concentrations of infused C-peptide do not affect basal insulin secretion or overall insulin secretory responses to oral or IV glucose. However, we cannot definitively exclude a small reduction in insulin secretion in the first hour after oral glucose ingestion.