Demonstration of C-peptide immunoreactivity in various body fluids and clinical evaluation of the determination of urinary C-peptide immunoreactivity.

C-peptide immunoreactivity (CPR) was demonstrated not only in plasma, but in urine, ascites, cerebrospinal fluid and pleural effusion. The concentration of CPR in urine was very high compared with that in the other body fluids and was easy to assay. CPR in urine after glucose administration or tolbutamide injection increased parallel to the change of CPR in plasma and also to that of IRI in normals or diabetics without renal disturbances. The result suggest that the determination of CPR in urine before and after stimulation of insulin secretion could serve as a simple indicator of insulin secretory function of pancreas.     

Responses of plasma insulin C-peptide and proinsulin-like components to glucose and glucagon in the pig.

This study was undertaken to evaluate the relative contribution of insulin, proinsulin-like components (PLC) and C-peptide toward plasma levels of immuno reactive insulin (IRI) and C-peptide immunoreactivity (CPR) in the pig and to elucidate the mode of secretion of PLC in the early phase of insulin release. Following the intravenous glucose loads, the concomitant secretion of CPR with that of IRI occured rapidly and the maximum plasma level of IRI was observed at an earlier time than that of CPR. Following the intravenous glucagon injection, the maximum plasma levels of IRI and CPR were observed at the same time in the early phase. After the gel filtration of acid alcohol extracts of plasma in a fasted state, a very small amount of PLC and a small amount of C-peptide as well as a small amount of insulin were detected. The results obtained from the gel filtration of extracts revealed that the increased amounts in IRI and CPR after the injection of glucose or glucagon consisted mostly and respectively of insulin and C-peptide in the pig, because the concentration of PLC increased only slightly in the early phase. In fact, plasma levels of CPR and IRI were essentially and respectively paralleled to those of insulin and C-peptide which were assayed after the gel filtration of extracts. In addition, the slight elevation of PLC in the early phase after these stimulations indicated that PLC was elicited into blood circulation at the same time of the secretion of insulin and C-peptide.     

Immunoreactive C-peptide in spontaneous syndromes of obesity and diabetes in mice

Immunoreactive C-peptide was evaluated in the plasma and pancreas of Aston ob/ob and C57BL/KsJ db/db mice in relation to disturbances in pancreatic B-cell function. At 18-24 weeks of age, ob/ob and db/db mice displayed hyperglycaemia (1.6 and 3.8 fold increases respectively) and hyperinsulinaemia (10.8 and 5.1 fold increases respectively) despite a similar pancreatic insulin content to their respective non-diabetic lean control mice. Immunoreactive C-peptide concentrations in the plasma and pancreas of the mutants corresponded with the degree of hyperinsulinaemia and pancreatic insulin content, and the insulin: C-peptide molar ratios in both mutants were similar to lean controls. In ob/ob mice parenteral glucose administration decreased plasma insulin and C-peptide concentrations, despite markedly raised glucose concentrations. However, administration of a low dose of insulin (5 U/kg) to lean mice and much higher doses of insulin (50 and 120 U/kg) to ob/ob mice markedly decreased plasma glucose and C-peptide concentrations. When the rate and extent of insulin-induced glucose suppression observed in ob/ob mice was mimicked in lean mice, an almost complete (95%) inhibition of C-peptide was achieved compared with a 57% decrease in the ob/ob mutant. Injection of ob/ob mice with glucose to counter the insulin-induced hypoglycaemia failed to affect the fall of C-peptide concentrations. The data suggest that the metabolic processing of insulin and C-peptide are undisturbed in obese-diabetic mice, and that the impaired suppression of circulating C-peptide by insulin-hypoglycaemia in ob/ob mice predominantly reflects impaired feedback inhibition by insulin.     

Failure of exogenous insulin to inhibit insulin secretion in man

In order to explore whether or not the negative feedback mechanism of insulin per se on insulin secretion exists in man, changes in plasma C-peptide immunoreactivity (CPR), as an index of pancreatic B cells secretory function, were studied in 6 nonobese healthy volunteers in the presence of high circulating levels of exogenous insulin. 10% glucose was infused concurrently so as to maintain blood sugar at the basal level. The insulin-glucose infusion was maintained for 120 minutes, achieving mean plasma levels of 140-180 mu1/ml. After this period, the insulin infusion was continued at the same rate for an additional 10 minutes while the glucose was omitted. Despite the elevated level of circulating insulin, no significant change in plasma CPR concentration was observed so long as the blood sugar was maintained at the basal levels. Following cessation of the glucose infusion, the plasma CPR levels declined with a decrease in blood sugar level. Under the conditions of the present study, no inhibitory effect of exogenous insulin on the secretory function of the B cells was noticed.     

Pancreatic B-cell function in non-insulin-dependent diabetes mellitus during successive periods of sulfonylurea and insulin treatment: serum C-peptide response to glucagon and urine C-peptide excretion

Serum C-peptide responses to glucagon and daily urine C-peptide excretion in successive periods of different treatment in two groups of patients with non-insulin-dependent diabetes mellitus (NIDDM) (mean interval between two tests less than 1 month) were compared. In group A patients (n = 8), the glycemic control was improved after transferring the treatment from sulfonylurea (SU) to insulin (fasting plasma glucose: SU: 192 +/- 47, insulin: 127 +/- 21 mg/dl, mean +/- S.D., p less than 0.01). Fasting serum C-peptide immunoreactivity (CPR) was significantly lower at the period of insulin treatment (SU: 1.93 +/- 1.01, insulin: 1.47 +/- 0.79 ng/ml, p less than 0.05), but there was no difference in the increase in serum CPR (maximal--fasting) (delta serum CPR) during glucagon stimulation in the two periods of treatment (SU: 1.70 +/- 0.72, insulin: 1.47 +/- 0.98 ng/ml). In group B patients (n = 7), there was no significant difference in glycemic control after transferring the treatment from insulin to SU (fasting plasma glucose: insulin: 127 +/- 24, SU: 103 +/- 13 mg/dl). Fasting serum CPR was significantly lower during the period of insulin treatment (insulin: 1.39 +/- 0.64, SU: 2.21 +/- 0.86 ng/ml, p less than 0.025), but delta serum CPR during glucagon stimulation still showed no significant difference between the two periods (insulin: 1.97 +/- 1.16, SU: 2.33 +/- 1.57 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Residual beta-cell function in type II diabetes and evaluation of the hepatic insulin extraction

Insulin and C-peptide (free insulin and C-peptide in insulin-treated patients) were measured after glucose stimulation in nine Type II diabetics on chlorpropamide, eleven insulin-treated maturity-onset diabetics and in 8 normal controls. Dissociation between C-peptide and insulin response to glucose was observed in several diabetics. The relation between incremental molar areas under C-peptide and insulin curves, after glucose challenge (delta CPR - delta IRI/delta CPR) were used to evaluate the hepatic insulin extraction in all but the insulin-treated diabetics. The lower insulin requirements and better control of the short-duration insulin-treated maturity-onset diabetics in relation to the long-term ones could not be explained either by the residual insulin secretion or by the level of "insulin antibodies". The chlorpropamide-responsive patients presented higher insulin levels after the glucose challenge and a lower hepatic insulin extraction than the non-responsive ones.     

Isolation and characterization of a trypsin-like protease from Trichoderma viride.

A serine endopeptidase with a molecular mass of 25 kDa has been purified from the culture filtrate of Trichoderma viride to electrophoretic homogeneity. The isoelectric point was determined at 7.3. Two carboxyl sites at Arg22 and Lys29 of the oxidized insulin B-chain were cleaved, and peptidyl-p-nitroanilide substrates with Lys or Arg at the P1 position were also hydrolyzed by the enzyme. These results suggest that the specificity of T. viride protease is similar to that of trypsin. However, the hydrolytic activity toward casein of T. viride protease was less than that of porcine trypsin. The amino-terminal sequence of the enzyme protein is similar to that of bovine trypsin. It seems that the trypsin of T. viride is a protease which is promising for the substitution of animal trypsin in the food industry and in medicine at this stage.     

Evidence of an insulin generated pyruvate dehydrogenase stimulating factor in rat brain plasma membranes

1. The results of this study indicates that the binding of insulin to brain plasma membranes activates a membrane protease which, by a trypsin like mechanism, produces a soluble factor that modulates the PDH behaviour when added to brain mitochondria. 2. The supernatant from brain plasma membranes incubated with 0.5 mg/ml trypsin added to mitochondria increases PDH activity levels and cancels PDH inhibition by NaF, as has already been seen when the plasma membranes are incubated with 25 microU/ml insulin. No such effects are obtained when the incubation is run out with 0.5 mg/ml chymotrypsin. 3. The supernatants from insulin or trypsin treated plasma membranes retain their activating properties on mitochondrial PDH also after dansylation; from these preparations a dansylated active on PDH material was separated by monodimensional chromatography on HPTLC silica Gel plates, using chloroform/1-butanol (93:7 v/v) as a solvent. 4. Insulin incubation of plasma membranes pretreated with protease inhibitors (leupeptin, phenylmethylsulfonylfluoride) or with exogenous trypsin, but not chymotrypsin substrates (esters of arginine and tyrosine) yields an inactive supernatant on PDH. 5. Insulin treated plasma membrane supernatants lose all stimulating properties on PDH after incubation for 1 hr with 2 mg/ml trypsin or chymotrypsin.     

Immunoreactive dog C-peptide level in the pancreatic vein

C-peptide immunoreactivity (CPR) levels were measured in dog superior pancreaticoduodenal vein using synthetic dog C-peptide and its antiserum. The basal CPR level was approximately twice as high as the basal immunoreactive insulin (IRI) level on a molar basis. Glucose (10 mg/kg/min) or arginine (250 mg/kg/min) infusion for 5 min into the superior pancreaticoduodenal artery caused a prompt, parallel increase in IRI and CPR. IRI and CPR were closely equimolar at peak secretions. One bolus administration of synthetic neurotensin (10 microgram/kg) into the same artery produced a mild hyperglycemic response and biphasic IRI and CPR responses at 30 min in the vein. The IRI and CPR increases were closely equimolar during the first phase of secretion, but during the second peak a larger increase was found in CPR than IRI. Upon infusion of synthetic substance P (50 ng/kg/min) for 30 min, IRI and CPR concentrations showed a parallel and closely equimolar fall. These results indicate that insulin and C-peptide were released from beta cells in equimolar concentrations.     

A proteolytic mechanism for the action of insulin via oligopeptide mediator formation

Evidence is presented that the chemical mediator of insulin action is a peptide(s) and most likely glycopeptide(s). The mediator is formed proteolytically because 1) protease inhibitors inhibit insulin action and 2) trypsin mimicks insulin action via mediator formation. Trypsin mediator does not faithfully reproduce the action of insulin mediator, which indicates that the sites of proteolytic cleavage by insulin and trypsin differ. A coordinated multivalent proteolytic mechanism by which insulin acts to trigger an external membrane-bound protease to cleave mediator from a membrane glycoprotein precursor is presented.     

C-peptide, insulin and proinsulinlike components in diabetic and nondiabetic human pancreas.

The contents of insulin and C-peptide extractable with acid alcohol from the tail of the pancreas and insulinoma were investigated, using gel filtration in seven nondiabetics including two patients with insulinoma and eight diabetics. The gel filtration patterns of both C-peptide and insulin in pancreatic extract were fairly stable even after the pancreas had been left for 14 hrs in the room temperature. In nondiabetics except cases of insulinoma the content of insulin in pancreas ranged from 1.42 to 4.56 U per gram and that of C-peptide from 8.76 to 25.63 microgram per gram wet pancreas. The proportion of proinsulinlike components (PLC) ranged from 0.01 to 2.04% of insulin plus PLC. In diabetics insulin content was low and ranged from 0 to 1.68 U per gram and that of C-peptide from 0 to 14.48 microgram per gram wet pancreas. In insulinoma, both insulin and C-peptide increased and PLC occupied 5.48 and 5.96%, respectively.     

Impaired insulin secretion in the neonatal rhesus monkey after chronic hyperinsulinemia in utero

The secretion of insulin by the pancreas of the newborn rhesus monkey that had been made experimentally hyperinsulinemic in utero was studied in 18 animals. Chronic in utero hyperinsulinemia was produced by the continuous subcutaneous delivery of 4.75 units of insulin per day for 18 +/- 1 days. After delivery, the insulin-containing pump was removed to allow neonatal insulin levels to drop to normal levels. By 6.5 +/- 1.0 hr after pump removal, plasma glucose, insulin, and C-peptide immunoreactivity (CPIR) were comparable in the control and experimental animals. At that point 300 micrograms of glucagon/kg body weight was given iv to stimulate insulin secretion. After 30 min a significant elevation (expressed as the percentage of basal levels) in plasma glucose by 250%, insulin by 200%, and CPIR by 200% was observed in the control animals. In contrast, no changes in plasma insulin or CPIR concentrations occurred, with an attenuated glucose response that was only one-fifth of the control response, in the experimental animals. These results along with the observed lowered concentrations of CPIR in the plasma and insulin in the pancreas at birth can be interpreted as evidence that insulin is an inhibitor of its synthesis and secretion in utero and that this abnormal intrauterine environment causes changes that persist into extrauterine life.     

Human plasma C-peptide immunoreactivity: its correlation with immunoreactive insulin in diabetes, and chronic liver and renal diseases.

The correlation between plasma C-peptide immunoreactivity (CPR) and immunoreactive insulin (IRI) was investigated during the oral glucose tolerance test in 20 normals, 127 diabetics, and 39 non-diabetics with chronic liver or renal disorders. When all subjects were included, the increment of CPR 30 minutes after glucose load (deltaCPR) correlated well with that of IRI (deltaIRI) (r = 0.66, p less than 0.001), but the return of CPR towards the basal level was delayed as compared with IRI. The positive correlation was also observed between the sum of 6 IRI and that of 6 CPR values during the glucose tolerance test in diabetics and controls (r = 0.53, p less than 0.001). deltaCPR/deltaBS (30 min.) was also well correlated with deltaIRI/deltaBS (30 min.), and was specifically low in diabetics. Insulin-treated maturity-onset diabetics showed low but considerable CPR responses while no CPR responses were observed in insulin-treated juvenile diabetics. In each plasma sample, CPR always exceeded IRI on the molar basis. At fasting CPR/IRI ratio was 15.6 +/- 1.7 (mean +/- SE) in normals and 14.9 +/- 1.3 approximately 16.9 +/- 1.0 in diabetics. In chronic liver diseases IRI response was augmented while CPR response was not different from that of controls, and the molar ratio of CPR/IRI was significantly low (9.5 +/- 1.1). On the contrary, it exceeded that of normals in chronic renal diseases (35.7 +/- 14.9). It is concluded that, first, the plasma CPR response appears to be a valuable indicator of pancreatic B-cell function, and second, it is, nevertheless, modified in chronic liver or renal disorders.     

Reduction of C-peptide secretion in noninsulin-dependent diabetic patients with long duration of insulin treatment

We examined the responses of serum free C-peptide immunoreactivity (CPR) during a 100 g oral glucose tolerance test (OGTT) on diabetic patients undergoing different kinds and durations of treatment. None of the patients were ketosis-prone or had any history of nephropathy and they all developed diabetes when over the age of 30. The sigma serum free CPR (the sum of serum free CPR values during OGTT) of group A (duration of insulin treatment was less than 5 years, N = 10) was found to be higher than that of group B (duration of insulin treatment was 5 years or more, N = 10) (p less than 0.005). On the other hand, the sigma serum free CPR of group C (treatment with an oral hypoglycemic agent for less than 5 years, N = 9) was not statistically different from that of group D (treatment with an oral hypoglycemic agent for 5 years or more, N = 11). There were no statistical differences between group A and group B in age at onset, duration of diabetes, daily insulin dose, relative body weight index, serum creatinine or sigma BG (the sum of blood glucose values during OGTT). Just before the start of insulin treatment, there were no significant differences between the two groups in the following: 1. fasting blood glucose values (all 10 patients measured in group A and 9 patients in group B) 2. blood glucose and plasma immunoreactive insulin (IRI) responses (7 patients measured in group A and 6 in group B). Among those with plasma IRI measured on the previous occasion, sigma serum free CPR was found to be higher in group A than in group B (p less than 0.025) at the time of the present study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Grape seed procyanidins improve β-cell functionality under lipotoxic conditions due to their lipid-lowering effect

Procyanidins have positive effects on glucose metabolism in conditions involving slightly disrupted glucose homeostasis, but it is not clear how procyanidins interact with β-cells. In this work, we evaluate the effects of procyanidins on β-cell functionality under an insulin-resistance condition. After 13 weeks of cafeteria diet, female Wistar rats were treated with 25 mg of grape seed procyanidin extract (GSPE)/kg of body weight (BW) for 30 days. To determine the possible mechanisms of action of procyanidins, INS-1E cells were separately incubated in high-glucose, high-insulin and high-oleate media to reproduce the conditions the β-cells were subjected to during the cafeteria diet feeding. In vivo experiments showed that chronic GSPE treatment decreased insulin production, since C-peptide levels and insulin protein levels in plasma were lower than those of cafeteria-fed rats, as were insulin and Pdx1 mRNA levels in the pancreas. GSPE effects observed in vivo were reproduced in INS-1E cells cultured with high oleate for 3 days. GSPE treatment significantly reduces triglyceride content in β-cells treated with high oleate and in the pancreas of cafeteria-fed rats. Moreover, gene expression analysis of the pancreas of cafeteria-fed rats revealed that procyanidins up-regulated the expression of Cpt1a and down-regulated the expression of lipid synthesis-related genes such as Fasn and Srebf1. Procyanidin treatment counteracted the decrease of AMPK protein levels after cafeteria treatment. Procyanidins cause a lack of triglyceride accumulation in β-cells. This counteracts its negative effects on insulin production, allowing for healthy levels of insulin production under hyperlipidemic conditions.     

C-Peptide Level in Fasting Plasma and Pooled Urine Predicts HbA1c after Hospitalization in Patients with Type 2 Diabetes Mellitus

In this study, we investigate how measures of insulin secretion and other clinical information affect long-term glycemic control in patients with type 2 diabetes mellitus. Between October 2012 and June 2014, we monitored 202 diabetes patients who were admitted to the hospital of Asahi Life Foundation for glycemic control, as well as for training and education in diabetes management. We measured glycated hemoglobin (HbA1c) six months after discharge to assess disease management. In univariate analysis, fasting plasma C-peptide immunoreactivity (F-CPR) and pooled urine CPR (U-CPR) were significantly associated with HbA1c, in contrast to ΔCPR and C-peptide index (CPI). This association was strongly independent of most other patient variables. In exploratory factor analysis, five underlying factors, namely insulin resistance, aging, sex differences, insulin secretion, and glycemic control, represented patient characteristics. In particular, insulin secretion and resistance strongly influenced F-CPR, while insulin secretion affected U-CPR. In conclusion, the data indicate that among patients with type 2 diabetes mellitus, F-CPR and U-CPR may predict improved glycemic control six months after hospitalization.     

Cold wind stimulation reflex.

The bradycardia induced by cold wind blown on the face and the early cephalic release of insulin induced by feeding have been shown to be caused by a vagal reflex stimulation. An experiment was designed to determine whether cold wind blown on the face would induce both pancreatic and cardiac stimulation. A 4 degrees C wind blown on the face for 4 min produced a rapid and persistent bradycardia, which interestingly persisted for up to 35 min after the test. The effect on respiration rate is more gradual and vanishes immediately after cold wind stimulation. Cold wind produced a slight reduction of insulin secretion, as evidenced by the fall of both plasma insulin and C-peptide, and caused a significant increase in plasma norepinephrine. These results suggest that the cold wind action of the vagus nerve is exerted on the heart and that of the sympathetic on the pancreas, whereas during the cephalic phase of feeding a vagal influence is observed on the pancreas and a sympathetic action on the heart. The mechanisms of the quantitative and qualitative control of these autonomic responses are not known and deserve further investigation.     

Insulin feedback actions: complex effects involving isoforms of islet nitric oxide synthase

The present study examined the effects of exogenous insulin on C-peptide release in relation to islet activities of neural constitutive nitric oxide synthase (ncNOS) and inducible NOS (iNOS). The dose-response curves for glucose-stimulated insulin and C-peptide release from isolated islets were practically identical: 0.05-0.1 nmol/l insulin stimulated, 1-100 nmol/l had no effect, whereas concentrations >/=250 nmol/l ("high insulin"), inhibited C-peptide release. Both the stimulatory and inhibitory effects were abolished by the phosphatidylinositol 3'-kinase inhibitor wortmannin. Addition of a NOS inhibitor partially reversed the inhibitory action of high insulin, but had no effect on the stimulatory action of low insulin (0.1 nmol/l). Moreover, high insulin markedly increased islet ncNOS activity and induced a strong iNOS activity. As shown biochemically and with confocal microscopy, the stimulatory action of high insulin on NOS activities and the associated inhibition of C-peptide release were reversed by raising cyclic AMP through addition of either glucagon-like peptide 1 (GLP-1) or dibutyryl cyclic AMP (Bt(2)cAMP) to the incubated islets. We conclude that the positive feedback mechanisms of action of insulin are independent of islet NOS activities and remain unclear. The negative feedback action of insulin, however, can be explained by its ability to stimulate both islet ncNOS activity and the expression and activity of iNOS. The effects on iNOS are most likely transduced through phosphatidylinositol 3'-kinase and are counteracted by raising islet cyclic AMP levels.     

Non-essential amino acids play an important role in adaptation of the rat exocrine pancreas to high nitrogen feeding

We have previously demonstrated that feeding a diet with a high amino acid (60% AA diet) content, as a mixture simulating casein, induced pancreatic growth and pancreatic protease production in rats. In the present study, we examined the effects of an increasing dietary content of essential amino acids (EAA, x1 - x3 in exp. 1 and x1 - x3.3 in exp. 2) and non-essential amino acids (NEAA, x1 - x3 in exp. 1 and x1 - x5.2 in exp. 2) on pancreatic growth, amylase and protease adaptation using casein-type amino acid mixtures (exp. 1, basal diet; 20% AA diet) and egg white-type amino acid mixtures (exp. 2, basal diet; 12% AA diet). Pancreatic growth and trypsin activity were induced as the dietary content of NEAA was increased in experiments 1 and 2. Amylase activity in the pancreas was also induced as the dietary content of NEAA was increased, even with the decrease in dietary carbohydrate in experiment 2. The values of all pancreatic variables decreased with the increase in dietary EAA (x2 and x3) without an increase in NEAA. The changes in the pancreas were coincident with increases in plasma arginine and lysine concentrations and a decrease in the plasma alanine concentration. In rats fed a 60% AA diet (EAA and NEAA x3), in the case of which the EAA content was balanced with the NEAA content, pancreatic growth and protease production increased and reached maximum levels as the plasma amino acid concentrations decreased, except for alanine. These results show that NEAA, not EAA, are associated with induction of pancreatic growth and protease production upon feeding a diet with a high AA content, and that some metabolites may be involved in the induction process. The suppression of pancreatic growth and protease production in rats fed the high EAA diets without balanced NEAA may be associated with impairment of amino acid metabolism rather than the increments in the concentration of one or more essential amino acids. Our results also suggest that there is an unknown mechanism or unknown factors involved in regulating pancreatic amylase.     

A fibrinolytic enzyme from a marine green alga, Codium latum

A fibrinolytic enzyme was isolated from a marine green alga, Codium latum, and designated C. latum protease (CLP). It also had fibrinogenolytic activity, hydrolyzing A alpha, B beta and gamma chains with preference in this order. As CLP hydrolyzed oxidized insulin B chain at position Arg22-Gly23, and the peptide map of lysozyme digested with CLP was similar to that with trypsin, CLP would be expected to have a high substrate specificity, similar to that of trypsin. Protease activity peaked at pH 10, and was completely inhibited by diisopropyl fluorophosphate (DFP). Therefore, we conclude that CLP is a trypsin-like serine protease.