Kinetics of human connecting peptide in normal and diabetic subjects.

The metabolic clearance rate (MCR) of synthetic human connecting peptide (C-peptide) was measured with a single-dose injection technique in six normal and seven diabetic subjects and with a constant infusion technique in one normal subject. The MCR of C-peptide did not differ in normal subjects (4.4 ml/min per kg; range, 3.7-4.9) and in diabetic subjects (4.7 ml/min per kg; range, 3.7-5.8). Employment of both techniques in one subject gave similar MCR. The average half-life of C-peptide in plasma calculated from the last 1-h period of the single-dose injection studies was longer in the insulin-dependent diabetics (42.5 min; range, 39.4-48.5) than in the normal subjects (33.5 min; range, 24.9-45.3). These results indicate that the beta-cell secretory capacity of normal and insulin-dependent diabetic subjects can be compared by measuring the C-peptide concentration in peripheral venous plasma. The difference in the half-life of C-peptide in plasma between diabetics and normals suggests an altered kinetics of the disappearance of the peptide, while the overall metabolism, as expressed by the MCR, is similar.

C-peptide in conditions other than diabetes mellitus.

Measurement of serum and urinary C-peptide has been shown to be of value in several conditions other than diabetes mellitus. It is particularly useful because it can distinguish endogenous beta cell secretion from exogenously administered insulin and because circulating insulin-binding antibodies do not interfere with its measurement. Because the liver removes little, if any, C-peptide, peripheral blood values may more accurately reflect beta cell secretion than do peripheral insulin levels. Clinically, serum C-peptide has been most useful in diagnosing hypoglycemic disorders. Diagnosis of insulinomas is facilitated in both diabetic and nondiabetic patients, and surreptitious insulin injection is readily detected. In studies of insulin regulation, circulating C-peptide has been used to demonstrate suppression of endogenous insulin secretion by exogenous insulin. Peripheral insulin and C-peptide levels have been compared in studies of the role of the liver in states of altered insulin homeostasis. Because of its higher urinary clearance, determination of urinary C-peptide is preferable to urinary insulin measurement in situations where frequent blood sampling is impossible or difficult to accomplish.

Determination of free and total insulin and C-peptide in insulin-treated diabetics.

Serum levels of free and total insulin as well as total C-peptide immunoreactivity (C-peptide and proinsulin) and C- peptide were measured in insulin-treated diabetics with circulating insulin antibodies by the addition of polyethylene glycol (PEG) before and after acidification. PEG resulted in complete precipitation of insulin antibodies from serum and made it possible to measure free insulin in the supernatant. Incubation of serum at 37 degrees C. for two hours before addition of PEG resulted in values for free insulin that probably resembled the in-vivo levels most closely. The same method could also be used to remove proinsulin bound to circulating insulin antibodies and permitted the measurement of C-peptide in the supernatant. Clinical studies using this approach indicate that combined measurements of serum free and total insulin and C-peptide provide information that is helpful in understanding the contribution of endogenous and exogenous insulin to the course and metabolic control of insulin-requiring diabetic patients.

Metabolic control in diabetic patients. Effect of insulin-secretory reserve (measured by plasma C-peptide levels) and circulating insulin antibodies.

We measured circulating hemoglobin A1 (HbA1) and fasting plasma C-peptide concentrations in 100 diabetic patients. Pancreatic insulin reserve showed a negative correlation with HbA1 concentrations in nonobese, insulin-treated patients but not in obese patients, whether they were treated with insulin, oral agent, or diet alone. Patients with fasting C-peptide concentrations above 0.1 pmol/ml had significantly better metabolic control than did those with lower values. Anti-insulin antibodies were measured in 37 patients. There was no correlation between metabolic control and the affinity constants or binding capacities of these antibodies.

A radioimmunoassay for circulating human proinsulin.

A radioimmunoassay for human proinsulin (hPl) has been developed using biosynthetic hPl prepared by recombinant DNA technology as immunogen, standard, and tracer. The antiserum was raised in a guinea pig and then adsorbed against insulin and C-peptide conjugated to Sepharose to improve its specificity. After adsorption of the antiserum, the cross-reactivities to insulin and C-peptide were each less than 0.2%. Competition studies using in vitro enzymatically split forms of proinsulin demonstrated that the major antigenic determinant recognized was the junctional region between the B-chain of insulin and the C-peptide. The range of the assay extended from 10 to 150 fmol/tube, with a 50% displacement of 45-55 fmol/tube. This sensitivity proved suitable for measurements of serum hPl concentrations during infusion of biosynthetic hPl into normal subjects and type I diabetic subjects. Eighty-five of 89 serum samples from the normal subjects and each of 20 samples from diabetic subjects diluted in parallel with the hPl standard. Since the direct assay sensitivity was not sufficient for measurement of endogenous hPl levels, a simple procedure for quantitative extraction of proinsulin-like material (PLM) from up to 40 ml of plasma on insulin antibody-Sepharose columns was developed. Logit-log slopes were calculated for dilutions of extracts of samples collected in the fasting state and 60 min after 75 g or oral glucose from eight healthy subjects. The slopes of 15 of the 16 samples did not differ significantly from the slope of the hPl standard.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of circulating human C-peptide.

Values reported for serum C-peptide immunoreactivity in healthy individuals vary considerably. To assess factors that contribute to this finding, three human C-peptide assay systems were developed utilizing three distinct antisera that react differently with various C-peptide fragments. Preparations of natural pancreatic and synthetic human C-peptide standards were compared immunologically in these systems. The curves of the natural C-peptide and the synthetic preparations were not identical. The relative immunoreactivity of each standard varied, depending on the particular antiserum used. Serum C-peptide concentrations varied when measured in the different assay systems. Furthermore, the results of dilution and recovery tests and stability of the C-peptide during storage showed differences among the three assays. Gel filtration of serum indicated heterogeneity within the major C-peptide peak, and in addition, a smaller peak of lower molecular weight material was present in some samples. Although degradation of serum C-peptide may occur during storage, fragments of C-peptide may also be secreted or arise during in-vivo metabolism. Thus, the present studies indicate the need for careful standardization and checking of each particular assay system before its use in clinical studies.

Relationship among peripheral glucose uptake, oxygen consumption, and glucose turnover in postabsorptive man.

To assess the importance of glucose uptake by muscle in determining total glucose utilization in the basal state, forearm glucose uptake (FGU), reflecting mainly skeletal muscle metabolism, and glucose turnover using [3-3H]glucose were studied simultaneously in 17 postabsorptive normal men. Mean +/- SE glucose disappearance was 2.36 +/- 0.14 mg/kg X min, amounting to 170 +/- 9 mg/min, while FGU was 0.049 +/- 0.009 mg/100 ml forearm X min. When the latter was calculated in terms of skeletal muscle in the body as a whole, muscle glucose utilization was found to be 24.7 +/- 4.5 mg/min, comprising only 13.5 +/- 1.9% of the total glucose disappearance. Forearm oxygen consumption was 6.6 +/- 0.5 mumol/100 ml forearm X min, of which only 26 +/- 5% could be accounted for by concurrent glucose uptake. These results suggest that in the basal state, glucose uptake by skeletal muscle accounts for 1) only a small percentage of total glucose disappearance and 2) only a minor proportion of peripheral oxygen consumption, which may be more dependent on lipid oxidation.

Gastric inhibitory polypeptide response to hyper- and hypoglycemia in insulin-dependent diabetics.

The response of gastric inhibitory polypeptide (GIP) levels to oral glucose in 11 insulin-dependent diabetics was compared to that in 8 age- and sex-matched healthy controls to determine whether they would show the pattern of GIP hypersecretion reported by other workers in maturity-onset, insulin-independent diabetes. One gram of glucose per kg bw resulted in a higher level of glycemia and a significantly diminished GIP response in diabetics when compared to controls (6,018 +/- 1,337 vs. 11,343 +/- 2,353 pg/ml.180 min min, respectively). There was virtually no beta cell response in the diabetics, as measured by changes in the levels of free insulin and connecting peptide. A significant lowering of glucagon levels occurred in the controls, while an inconsistent response was seen in the diabetics. An insulin infusion test was administered to test the hypothesis that insulin suppresses GIP secretion. Although hyperinsulinism, hypoglycemia, and suppression of endogenous insulin secretion were produced in the controls, no suppression of baseline GIP was detected. Similarly, hyperinsulinism and hypoglycemia failed to suppress baseline GIP levels in the diabetics. These results do not support a direct role for insulin in suppressing GIP in normal or diabetic subjects.

The metabolic effects of biosynthetic human proinsulin in individuals with type I diabetes.

To assess the significance of deficiency of circulating proinsulin in patients with type I diabetes mellitus, we studied the metabolic effects of biosynthetic human proinsulin in 24 patients. After withdrawing insulin, an infusion of proinsulin to physiological plasma levels did not prevent elevations of plasma glucose or beta-hydroxybutyrate. During steady state infusions of insulin and proinsulin, 13.7 times the steady state plasma level of proinsulin compared to insulin was required to maintain euglycemia. This finding indicates that proinsulin is approximately 7.3% as biologically active as insulin on a molar basis in maintaining glucose control. The MCRs of insulin and proinsulin during these steady state infusions were 12.5 +/- 2.2 (+/- SEM) and 2.62 +/- 0.33 ml/kg X min, respectively. After maintaining euglycemia overnight with an infusion of insulin or proinsulin and then acutely stopping these infusions, the rise in plasma glucose after proinsulin was delayed significantly compared to insulin, consistent with proinsulin's slower clearance. Further studies are necessary to determine whether biosynthetic human proinsulin has a specific role in the treatment of diabetes.

C-Peptide and insulin secretion in Pima Indians and Caucasians: constant fractional hepatic extraction over a wide range of insulin concentrations and in obesity.

Peripheral serum insulin and C-peptide concentrations during oral glucose tolerance tests were measured in 10 nondiabetic Pima Indians and 10 nondiabetic Caucasians with varying degrees of obesity. Although both insulin and C-peptide levels were elevated in the Indians compared to the Caucasians (p less than 0.05), hepatic insulin extraction, measured by comparing the C-peptide to insulin ratios, was similar over a wide range of insulin concentrations in both groups. The ratios of C-peptide to insulin were independent of the degree of obesity. These studies indicate that the peripheral hyperinsulinemia in Pima Indians and obese subjects is due in general to pancreatic hypersecretion rather than to diminished hepatic extraction of insulin.

The influence of graded hyperglycemia with and without physiological hyperinsulinemia on forearm glucose uptake and other metabolic responses in man.

We studied the influence of hyperglycemia on glucose homeostasis in man by determining the effect of graded hyperglycemia on peripheral glucose uptake and systemic metabolism in the presence of basal and increased serum insulin concentrations in 10 normal men. This was achieved by the simultaneous application of forearm and clamp techniques (euglycemic and hyperglycemic) during the combined iv infusion of somatostatin, glucagon, and insulin. While mean (+/- SE) basal serum insulin levels (14 +/- 2 microU/ml) were maintained, the elevation of fasting arterial glucose concentrations (90 +/- 1 mg/dl) to 146 +/- 1 and 202 +/- 1 mg/dl (each for 120 min) increased forearm glucose uptake (FGU) only modestly from 0.06 +/- 0.01 to 0.15 +/- 0.02 and then to 0.24 +/- 0.03 mg/100 ml forearm X min, respectively. During physiological hyperinsulinemia (47 +/- 3 microU/ml), the influence of similar graded hyperglycemia on FGU was considerably enhanced. At plasma glucose concentrations of 90 +/- 1, 139 +/- 1, and 206 +/- 1 mg/dl, FGU rose to 0.33 +/- 0.05, 0.59 +/- 0.07, and 0.83 +/- 0.12 mg/100 ml forearm X min, respectively. The glucose infusion rate required to maintain the glucose clamp with basal insulin levels was 1.08 +/- 0.20 and 2.67 +/- 0.39 mg/kg X min at glucose concentrations of 146 +/- 1 and 202 +/- 1 mg/dl, respectively. During physiological hyperinsulinemia, however, the glucose infusion rate required was 4.15 +/- 0.39, 9.45 +/- 1.05, and 12.70 +/- 0.81 mg/kg X min at glucose levels of 90 +/- 1, 139 +/- 1, and 206 +/- 1 mg/dl, respectively. Lactate concentrations rose significantly during hyperglycemia, but the rise in the presence of increased insulin concentrations (from 0.72 +/- 0.06 to 1.31 +/- 0.11 mmol/liter; P less than 0.001) considerably exceeded the increment (from 0.74 +/- 0.05 to 0.92 +/- 0.03 mmol/liter) with basal insulin levels. While both FFA and glycerol concentrations were immediately reduced by euglycemic hyperinsulinemia, the fall in FFA during hyperglycemia in the presence of basal insulin levels preceded the decrease in glycerol concentrations by 45 min. Forearm oxygen consumption did not change throughout the study.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneity of circulating C-peptide.

Serum C-peptide levels vary when measured with different immunoassay systems. In order to assess the factors contributing to this finding, serum C-peptide was measured in two assays utilizing different antisera, but the same standards and labeled peptide. The antisera were characterized with synthetic C-peptide fragments and their reactivities towards some of these fragments differed. The results of dilution and recovery tests and stability of the C-peptide during storage showed differences between the two assays. Gel filtration experiments indicated heterogeneity within the major C-peptide peak, and, in addition, a smaller peak of lower molecular weight material was present in some sera. Although degradation of serum C-peptide may occur during storage or with freezing and thawing, fragments of C-peptide may also be secreted or arise during in vivo metabolism.

Peripheral lactate and oxygen metabolism in man: the influence of oral glucose loading.

We investigated the influence of oral glucose loading (100 g) on glucose, lactate, and oxygen metabolism by deep (mainly muscle) and superficial (mainly skin and adipose tissue) forearm tissues. In normal men aged 19 to 32 years (mean +/- SE, 24 +/- 1), basal arterialized venous-deep venous (A-DV) and arterialized venous-superficial venous (A-SV) plasma glucose concentration differences were 4.1 +/- 1.0 (P less than 0.001) and 4.7 +/- 1.0 (P less than 0.005) mg/dL, respectively, but increased markedly following glucose loading. During the first, second, and third hours after glucose ingestion, A-DV differences were 54 +/- 6,43 +/- 3, and 20 +/- 4 mg/dL, respectively, while the corresponding A-SV differences were 39 +/- 4, 17 +/- 2, and 8 +/- 2 mg/dL, respectively. Forearm glucose uptake by deep (FGU-D) and superficial (FGU-S) tissues basally was 0.057 +/- 0.010 and 0.012 +/- 0.002 mg/100 mL forearm/min respectively. From 15 to 180 minutes after glucose loading, mean FGU-D and FGU-S rose to 0.524 +/- 0.083 and 0.056 +/- 0.006 mg/100 mL forearm/min, respectively. Basal A, SV, and DV lactate concentrations were 0.55 +/- 0.04, 0.78 +/- 0.03, and 0.57 +/- 0.04 mmol/L, respectively (A-SV, P less than 0.001; SV-DV, P less than 0.001; A-DV, NS). Lactate production by superficial tissues (0.079 +/- 0.015 mumol/100 mL forearm/min) accounted for 62% of concurrent FGU-S.(ABSTRACT TRUNCATED AT 250 WORDS)

Basal glucose homeostasis with and without fixed concentrations of glucagon and insulin.

The aim of this study was to investigate the extent to which the basal steady state could be maintained with fixed concentrations of glucagon and insulin. To this end, arterial plasma glucose concentrations and peripheral glucose uptake (using the forearm technique) were compared in healthy men (age 19 to 23 years) in the normal postabsorptive state and after suppression of endogenous pancreatic secretion. Two groups (A and B), each consisting of four men, were studied. In group A, the study comprised a control period (I) of 40 minutes followed by a test period (II) of 180 minutes during which normal pancreatic secretion was maintained throughout. In group B, the study comprised a control period (I) of 40 minutes, a stabilization period (II) of 120 minutes, and a test period (III) of 120 minutes. After the control period with normal pancreatic secretion, a new steady state with fixed hormone concentrations was established during the first 90 minutes of period II using simultaneous infusions of somatostatin (250 micrograms/h), insulin (0.15 mU/kg/min) and glucagon, the latter being adjusted to maintain a stable arterial glucose level similar to the preceding control concentration. Thereafter, without further adjustment of the glucagon infusion rate, observations were continued during period III to assess the maintenance of the steady state. In group A, the range of variation in arterial glucose concentrations during periods I and II was 4.0 +/- 0.9 and 6.5 +/- 1.3 mg/dL, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of peripheral glucose uptake after oral glucose loading and a mixed meal.

Forearm glucose uptake (FGU) and other metabolic responses were studied in six normal men for three hours after a 75-g oral glucose load and a mixed meal containing 75 g carbohydrate. After the meal the rise in arterial glucose levels was considerably less than that following the oral glucose load but the overall insulin responses from 0 to 180 minutes were not statistically different. Although the initial rise in FGU was more gradual after the meal, the subsequent elevation was more sustained and, at the termination of the study, exceeded significantly that seen after the oral glucose load. The rise in GIP levels during the first hour was similar after the meal and the oral glucose load, but thereafter concentrations following the oral glucose load fell while those after the meal continued to rise. When the incremental area (delta) is used as the index of response, the results show that while the glucose response (delta G) after the meal (19.1 +/- 5.3 units) was only 26% of that after oral glucose loading (72.7 +/- 7.0 units), the corresponding increase in FGU (delta FGU) reached 62% (55.0 +/- 12.8 units after the meal, 89.2 +/- 20.0 units after the oral glucose load). Thus, the increase in peripheral glucose uptake relative to the glycemic response (delta FGU/delta G) was significantly greater after the meal than following the oral glucose load alone (P less than 0.05). In conclusion, relative to the rise in arterial glucose levels, peripheral glucose uptake is greater after a meal than after glucose loading with an equivalent carbohydrate challenge. Furthermore, the present data support previous studies emphasizing the failure of GIP alone to explain the entero insular axis.

Good diabetic control early in pregnancy and favorable fetal outcome.

This study of 74 diabetic pregnant women shows that tight maternal blood glucose control before the 32nd week of gestation significantly reduces the incidence of fetal macrosomia (11%) when compared with that of patients with fair to poor control before the 32nd week of gestation (44%, P less than .05) or with those whose good diabetic control was not achieved until after the 32nd week of gestation (34%, P less than .05). The macrosomic infant produced by a diabetic mother is associated frequently with an elevated amniotic fluid C-peptide level, which shows the evidence of intrauterine fetal hyperinsulinism. The use of tight diabetic control early in pregnancy to reduce the risk of fetal macrosomia and/or neonatal complications is of clinical importance in the management of diabetes in pregnancy.

Specific cytosol binding of diethylstilbestrol in rat skeletal muscle.

Rat skeletal muscle cytosol proteins bound 3H-diethylstilbestrol (3H-DES). More than 90% of this binding was high capacity and low affinity. Serum albumin accounted for roughly 50-60% of the binding, as evidenced by its precipitation with anti-rat albumin IgG. About half of the binding was distinguishable from albumin and other serum proteins by its precipitation in 40% saturated ammonium sulfate. This material sedimented at 4-5S in high-salt sucrose gradients, and resolved into two components (8S and 4-5S) in low-salt. Following incubation at 23-27 degree C for one hour, 2% of the bound 3H-DES in whole cytosol (approximately 2 fmole/Mg cytosol protein) was retained by DNA cellulose, and was eluted with 0.6 M KCl. This small fraction of the total binding was inhibited by estrogens and DES analogues: estradiol-17 beta, DES, dienestrol, and hexestrol were strong inhibitors; isodienestrol, dimethylstilbestrol, estradiol-17 alpha, estrone, tamoxifen, MER-25, CI-628, and nafoxidine were weak inhibitors; dihydrotestosterone, testosterone, and prednisone did not compete. These observations indicate that specific estrogen-binding sites exist in rat skeletal muscle.