William Pugh

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.

Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice.

Mutations in the gene for the transcription factor hepatocyte nuclear factor (HNF) 1alpha cause maturity-onset diabetes of the young (MODY) 3, a form of diabetes that results from defects in insulin secretion. Since the nature of these defects has not been defined, we compared insulin secretory function in heterozygous [HNF-1alpha (+/-)] or homozygous [HNF-1alpha (-/-)] mice with null mutations in the HNF-1alpha gene with their wild-type littermates [HNF-1alpha (+/+)]. Blood glucose concentrations were similar in HNF-1alpha (+/+) and (+/-) mice (7.8+/-0.2 and 7.9+/-0.3 mM), but were significantly higher in the HNF-1alpha (-/-) mice (13.1+/-0.7 mM, P < 0.001). Insulin secretory responses to glucose and arginine in the perfused pancreas and perifused islets from HNF-1alpha (-/-) mice were < 15% of the values in the other two groups and were associated with similar reductions in intracellular Ca2+ responses. These defects were not due to a decrease in glucokinase or insulin gene transcription. beta cell mass adjusted for body weight was not reduced in the (-/-) animals, although pancreatic insulin content adjusted for pancreas weight was slightly lower (0.06+/-0.01 vs. 0.10+/-0.01 microg/mg, P < 0.01) than in the (+/+) animals. In summary, a null mutation in the HNF-1alpha gene in homozygous mice leads to diabetes due to alterations in the pathways that regulate beta cell responses to secretagogues including glucose and arginine. These results provide further evidence in support of a key role for HNF-1alpha in the maintenance of normal beta cell function.

Metabolism of C-peptide in the dog. In vivo demonstration of the absence of hepatic extraction.

The in vivo hepatic metabolism of connecting peptide (C-peptide) in relation to that of insulin has not been adequately characterized. A radioimmunoassay for dog C-peptide was therefore developed and its metabolism studied in conscious mongrel dogs, with sampling catheters chronically implanted in their portal and hepatic veins and femoral artery. The hepatic extraction of endogenous C-peptide under basal conditions was negligible (4.3 +/- 4.5%) and was similar to the hepatic extraction of C-peptide measured during the constant exogenous infusion of C-peptide isolated from dog pancreas. Simultaneously measured hepatic extraction of endogenous and exogenously infused insulin were 43.8 +/- 7.6 and 47.5 +/- 4.4%, respectively. The metabolic clearance rate of infused C-peptide was 11.5 +/- 0.8 ml/kg per min and was constant over the concentration range usually encountered under physiological conditions. In additional experiments, the effect of parenteral glucose administration on the hepatic extraction of C-peptide and insulin was investigated. The hepatic extraction of C-peptide (6.2 +/- 4.0%) was again negligible in comparison with that of insulin (46.7 +/- 3.4%). Parenteral glucose administration did not affect the hepatic extraction of either peptide irrespective of whether it was infused peripherally, intraportally, or together with an intraportal infusion of gastrointestinal inhibitory polypeptide. The fasting C-peptide insulin molar ratio in both the portal vein (1.2 +/- 0.1) and femoral artery (2.1 +/- 0.3) was also unaffected by the glucose stimulus. These results therefore indicate that, since the hepatic extraction of C-peptide is negligible and its clearance kinetics linear, the peripheral C-peptide concentration should accurately reflect the rate of insulin secretion. New approaches to the quantitation of hepatic extraction and secretion of insulin by noninvasive techniques are now feasible.

The Limitations to and Valid Use of C-Peptide as a Marker of the Secretion of Insulin

The accuracy with which the secretion rate of insulin can be calculated from peripheral concentrations of C-peptide was investigated in conscious mongrel dogs. Biosynthetic human C-peptide and insulin were infused intraportally and their concentrations measured in the femoral artery. During steady-state infusions of C-peptide, the peripheral concentration changed in proportion to the infusion rate and the metabolic clearance rate (5.2 ± 0.3 ml/kg/min) remained constant over a wide range of plasma concentrations. Application of a two-compartment mathematical model, in which the model parameters were estimated from analysis of C-peptide decay curves after intravenous bolus injections, allowed the intraportal infusion rate of C-peptide to be derived from peripheral C-peptide concentrations, even under non-steady-state conditions. Estimates of the intraportal infusion rate based on this model were 102.4 ± 2.6% of the actual infusion rate as it was increasing and 102.3 ± 5.5% of this rate as it was falling. The peripheral C-peptide : insulin molar ratio was influenced by the rate at which equimolar intraportal infusions of C-peptide and insulin were changed. The baseline C-peptide : insulin molar ratio (4.1 ± 0.9) increased to peak values of 8.2 ± 0.6,10.3 ± 2.0, and 14.9 ±1.3 when the infusion rate was increased and then decreased rapidly. Peak values of only 5.7 ±1.2 were found if the intraportal infusion rate was changed slowly. In conclusion: (1) under steady-state conditions the secretion rate of insulin can be calculated as the product of the peripheral concentration of C-peptide and its MCR; (2) under non-steady-state conditions, however, application of more complex mathematical models, such as the two-compartment model used in the present study, allows insulin secretion rates to be accurately calculated at discrete time points; and (3) under non-steady-state conditions the C-peptide:insulin molar ratio is influenced not only by the extent of hepatic insulin extraction but by other factors, including the rate of change of insulin secretion and the clearance rate of C-peptide. Changes in this ratio should therefore not be assumed to reflect changes in hepatic insulin extraction.

Philadelphia chromosome-negative chronic myelogenous leukaemia: a morphological reassessment

Morphological re‐examination of 25 cases classified as Ph1‐negative chronic myelogenous leukaemia (CML), with particular attention to the recent French‐American‐British (FAB) group proposals for the diagnosis of the myelodysplastic syndromes, led to reclassification of all but one of these cases. Upon review of pretreatment material, seven of the 25 cases (28%) were considered to represent myeloproliferative and reactive conditions other than CML. Of the remaining 18 cases, 17 (94%) were reclassified as myelodysplastic syndromes (MDS). These included one case of refractory anaemia (RA), three of refractory anaemia with excess of blasts (RAEB), four of RAEB in transformation (RAEBT), and nine of chronic myelomonocytic leukaemia (CMMoL). Haematological findings in these 17 patients were compared to those in 50 randomly selected patients with Ph1‐positive CML. Whereas an absolute basophilia in the peripheral blood was a nearly constant feature in the Ph1‐positive group, it was lacking in all but one of the patients with myelodysplastic syndromes. Differences in the pattern and degree of dysplasia were also noted. Only one of the 25 cases studied was considered morphologically and clinically indistinguishable from Ph1‐positive CML. It appears that Ph1‐negative CML constitutes a heterogeneous group of prognostically distinct disorders and the, in most instances, careful morphologic examination will permit precise classification.

C-peptide and insulin secretion. Relationship between peripheral concentrations of C-peptide and insulin and their secretion rates in the dog.

Estimation of the insulin secretory rate from peripheral C-peptide concentrations depends upon the following characteristics of C-peptide kinetics: (a) equimolar secretion of insulin and C-peptide by pancreatic beta cells; (b) negligible hepatic extraction of C-peptide; (c) constant metabolic clearance rate (MCR) of C-peptide over a physiological and pathophysiological range of plasma levels; and (d) proportional changes in the secretion rate of C-peptide and its peripheral concentrations under varying physiological conditions. In the present experiments, the relationship between a variable intraportal infusion of C-peptide and its concentration in the femoral artery was explored in 12 pancreatectomized dogs. As the infusion of C-peptide was rapidly increased, the magnitude of its peripheral concentration initially increased less than the infusion rate by 20-30%. After an equilibration period of approximately 30 min, however, further increases and decreases in the intraportal infusion were accompanied by nearly proportional changes in its peripheral concentration. Estimates of the amount of C-peptide infused during the experiment based on the steady state C-peptide MCR and its peripheral concentration were within 20% of the amount of C-peptide actually infused. These experiments demonstrate that the portal delivery rate of C-peptide can be calculated from its MCR and peripheral concentration in the dog. They also provide a basis for testing the validity of more complicated models of insulin secretion based on peripheral C-peptide concentrations in the dog as well as other species, including man. Finally, we have shown that the hepatic extraction of endogenously secreted C-peptide is negligible in the basal state (3.1 +/- 6.1%), and does not change after oral glucose ingestion. The MCR of exogenous dog C-peptide was similar whether measured by constant peripheral intravenous infusion (12.3 +/- 0.7 ml/kg per min), constant intraportal infusion (13.4 +/- 0.6 ml/kg per min), or analysis of the decay curve after a bolus injection (13.5 +/- 0.7 ml/kg per min).

Expression of Calcium Channel mRNAs in Rat Pancreatic Islets and Downregulation After Glucose Infusion

Recent studies have shown that two different voltage-dependent Ca2+ channels are expressed in pancreatic islets, the β-cell/neuroendocrine-brain and the cardiac subtypes. The effects of chronic hyperglycemia on the levels in pancreatic islets of the mRNAs encoding the α1-subunits of the β-cell and cardiac subtype Ca2+ channels were studied in rats made hyperglycemic by infusion of glucose for 48 h. A competitive reverse transcriptase-polymerase chain reaction procedure was used to obtain quantitative data on the levels of these two transcripts in islets obtained from individual rats. The quantitative polymerase chain reaction data indicate that the levels of mRNA encoding the α 1-subunit of the β-cell Ca2+ channel are 2.5-fold > those for the cardiac subtype. The levels of β-cell Ca2+ channel mRNA were 72.9% lower in the glucose-infused animals when compared with the saline-infused animals (P < 0.005) and those of the cardiac channel were 72.1% lower in the animals infused with glucose (P < 0.02). In contrast, glucose infusion resulted in a twofold increase in insulin mRNA levels and did not significantly alter levels of β-actin mRNA. In situ hybridization studies revealed that the mRNAs for these two Ca2+ channels are expressed at higher levels in normal rat islets than in the surrounding acinar tissue, which suggests that the observed changes in mRNA levels occur within cells of the pancreatic islet. To assess the possible functional consequences of this reduction in expression of mRNA for the Ca2+ channels, the insulin secretory responses of perfused pancreases to the Ca2+ channel agonist Bay K8644 were studied. In pancreases from glucose-infused animals, the relative incremental insulin secretory response to Bay K8644 was reduced, and a similar blunting of the acute response to glucose also was seen. In summary, the predominant voltage-dependent Ca2+ channels subtype expressed in islets under basal conditions and after glucose infusion is the β-cell form with lower levels of the cardiac subtype. Glucose infusion for 48 h results in a significant reduction in the mRNA levels for both the β-cell and cardiac subtypes, and this is associated with enhanced basal secretion but reduced responses to glucose and the Ca2+ channel agonist Bay K8644.

Equilibrium Binding Assay and Kinetic Characterization of Insulin Antibodies

A simple and precise equilibrium binding assay has been developed for the quantitation of circulating insulin antibodies. This procedure differs from previously reported methods in that (1) serum samples were deinsulinized in order to avoid endogenous insulins interfering with the binding assay, (2) l25I-monoiodoinsulin was used to prevent artifacts resulting from variability in ligand binding due to excessive iodination, and (3) separation of free and bound insulin was accomplished by rapid precipitation of the hormone-antibody complexes without disturbing the binding equilibrium. This approach prevented dissociation and underestimation of the bound species. In agreement with previous reports, two classes of antibody sites (high-affinity-low-capacity and low-affinity-high-capacity) were demonstrated in the sera of insulin-treated diabetic patients. No consistent differences existed in the association constants and/or binding capacities for insulins of bovine, porcine, and human origin, even though the immunogenic stimulus for antibody formation in these patients was treatment with commercial preparations of bovine and porcine insulins. Thus, higher affinities and/or binding capacities for human versus bovine and porcine insulins does not provide evidence for an autoimmune origin of insulin antibodies, as has been previously proposed. Significant differences in the antibody-binding capacities for all three species of insulin were found in individual serum samples, and underestimation of antibody titers may occur if only one species of the hormone is used. Kinetic studies on the dissociation of the insulin-antibody complexes revealed two classes of dissociating sites, in agreement with the equilibrium binding data. The corresponding dissociation rate constants were large enough to implicate the release of insulin from the pool of antibody-bound hormone as a possible hypoglycemic mechanism in some diabetic patients.

Basal insulin hypersecretion in insulin-resistant Zucker diabetic and Zucker fatty rats: Role of enhanced fuel metabolism

The biochemical mechanisms responsible for basal hyperinsulinemia in insulin-resistant states have not been fully defined. We therefore studied pancreatic beta-cell function in vitro to characterize the relative importance of fuel metabolism or secretion via a constitutive pathway in the maintenance of high basal insulin secretion in Zucker diabetic fatty (ZDF) and Zucker fatty (ZF) rats. Insulin secretion from ZF (10+/-1.8 v 5+/-0.6 pmol/ng DNA/h) and ZDF (30+/-4 v 7+/-0.8 pmol/ng DNA/h) islets at 2.8 mmol/L glucose was two to four times greater than secretion from islets of lean littermate control rats. In response to a decreasing glucose concentration (from 12 to 0 mmol/L), a paradoxical increase in insulin secretion was observed in perfused ZDF rat pancreas. Insulin secretion at 2.8 mmol/L glucose was suppressed approximately 70% to 80% in islets from ZDF and ZF rats following exposure to diazoxide, a K+-adenosine triphosphate (K(ATP)) channel opener that inhibits membrane depolarization, or rotenone and oligomycin, agents that inhibit ATP production, or by incubation at 23 degrees C. Inhibition of glycolysis with mannoheptulose, 2-deoxyglucose, and iodoacetate or fatty acid oxidation with a carnitine palmitoyltransferase I inhibitor also significantly inhibited basal insulin secretion in islets of ZDF and ZF rats but not their lean littermates. Furthermore, the glycolytic flux at 2.8 mmol/L glucose was significantly higher in ZDF islets versus ZDF lean littermate (ZLC) islets (2.2+/-0.1 v 3.7+/-0.3 pmol/ng DNA/2 h, P < .01) and was suppressed by mannoheptulose. In ZDF and ZF islets, high basal insulin secretion was maintained despite a 50% reduction in the rate of proinsulin/insulin biosynthesis at 2.8 mmol/L glucose. The rate of proinsulin to insulin conversion and the ratio of proinsulin to insulin secretion by islets of ZDF rats were similar to the values in the lean littermates. Thus, basal hypersecretion in these two insulin-resistant models appears to be related to enhanced fuel metabolism rather than the contribution of a constitutive pathway of secretion.

Relationship between decrements in glucose level and metabolic response to hypoglycemia in absence of counterregulatory hormones in the conscious dog.

To determine the relationship between decreases in glucose and metabolic regulation in the absence of counterregulatory hormones, we infused overnight-fasted, conscious, adrenalectomized dogs (lacking cortisol and EPI) with somatostatin (to eliminate glucagon and growth hormone) and intraportal insulin (30 pmol · kg−1 · min−1), creating arterial insulin levels of ∼2000 pM. Glucose was infused during one 120-min period, two 90-min periods, and one 45-min period to establish levels of 5.9 ± 0.1, 3.4 ± 0.1, 2.5 ± 0.1, and 1.7 ± 0.1 mM, respectively. NE levels were 1.24 ± 0.23, 1.85 ± 0.27, 2.04 ± 0.26, and 2.50 ± 0.20 nM, respectively. During the euglycemic control period, the liver took up glucose (7.5 ± 1.9 μmol · kg−1 · min−1), but hypoglycemia triggered successively greater rates of net hepatic glucose output (3.0 ± 0.7, 4.6 ± 0.9, and 6.9 ± 1.4 μmol · kg−1 · min−1). Total gluconeogenic precursor uptake by the liver increased with hypoglycemia. Intrahepatic gluconeogenic efficiency rose progressively (by 106 ± 42,199 ± 56, and 268 ± 55%). Both glycerol and NEFA levels rose, indicating lipolysis was enhanced. Net hepatic NEFA uptake and ketone production increased proportionally, but the ketone level rose only with severe hypoglycemia. In conclusion, despite marked hyperinsulinemia and the absence of glucagon, EPI, and cortisol, we observed that lipolysis and glucose and ketone production increase in response to decreases in glucose. This suggests that neural and/or autoregulatory mechanisms can play a role in combating hypoglycemia.