Free C-peptide Research Articles

The comparison of different quantitative criteria of the Dawn Phenomenon and its impact on blood glucose fluctuation in type 2 diabetes

A total of 128 individuals with type 2 diabetes underwent continuous glucose monitoring for 3 consecutive days. The dawn phenomenon was defined by three different parameters according to the previous research: (1)the absolute increase of glucose level from nocturnal nadir to prebreakfast value(∂G) above 20 mg/dl; (2)∂G above 10 mg/dl; (3)insulin requirement increased at least 20%. The participants were secondarily separated by presence/absence of a dawn phenomenon based on the definitions above. The impact on blood glucose fluctuation of different groups was assessed according to the standard deviation of blood glucose(SDBG), the area under curve above 10 mmol/L(AUC), and the mean amplitude of glycemic excursions(MAGE), etc. The frequencies of dawn phenomenon were 64.8%(∂G≥20mg/dl), 85.2%(∂G≥10 mg/dl), and 59.4%(rise in insulin requirement≥20%)respectively. The impacts on SDBG, AUC, MAGE, and MODD were without statistical difference(P>0.05) between the presence and absence of the dawn phenomenon patients when ∂G≥10 mg/dl. However, the differences reached statistical significance(P<0.05) when ∂G≥20 mg/dl and the increase in insulin requirement≥20%. Besides, the incidence of dawn phenomenon was positively correlated with HOMA-IR, HbA1C, and free C-peptide. Dawn phenomenon is a very frequent event in type 2 diabetes and not only impacts the overall glycemic control but also exaggerates glucose fluctuation. To be clinically relevant, ∂G≥20mg/dl should be taken as the quantitative criterion of the dawn phenomenon. (Chin J Endocrinol Metab, 2016, 32: 117-120) Key words: Diabetes mellitus, type 2; Dawn phenomenon; Incidence; Continuous glucose monitoring system; Blood glucose fluctuation

Directed overexpression of insulin in Leydig cells causes a progressive loss of germ cells

The primary goal of this study was to determine the 5'region of the Insl3 gene that specifically targets the expression of human insulin to Leydig cells, and to explore whether the testicular proinsulin is efficiently processed to insulin that is able to rescue the diabetes in different mouse models of diabetes. We show here that the sequence between nucleotides -690 and +4 of mouse Insl3 promoter is sufficient to direct the Leydig cell-specific expression of the human insulin transgene (Insl3-hIns). We also found that the 3'untranslated region (3'UTR) of Insl3 was effective in enhancing transgene expression of the insulin in vivo. Expression analysis revealed that the temporal expression pattern of the hIns transgene in Leydig cells of transgenic testes is roughly the same as that of the endogenous Insl3. Despite the Leydig cells translate human proinsulin and secrete a significant level of free C-peptide into the serum, the Leydig cell-derived insulin is not able to overcome the diabetes in different mouse models of diabetes, suggesting a lack of glucose sensing mechanisms in the Leydig cells. A consequence of overexpression of the human proinsulin in Leydig cells was the decrease of fertility of transgenic males at older ages. Germ cells in transgenic males were able to initiate and complete spermatogenesis. However, there was a progressive and age-dependent degeneration of the germ cells that lead to male infertility with increasing age.

The utility of the C‐peptide in the phenotyping of patients candidates for pancreas transplantation

It is not unusual for simultaneous pancreas and kidney transplantation (SPK) to be performed in patients with type 2 diabetes (T2D), clinically classified as having type 1 diabetes (T1D). C-peptide determination is useful to identify these patients. We describe the prevalence and characteristics of patients with C-peptide levels >3 ng/mL, classified with T2D in 172 patients referred for SPK from 1998-2006. Nine patients (5.2%) fulfilled this criteria (mean free C-peptide 9.08 ng/mL) and were older at diabetes onset (23.5 vs. 12 yr, p < 0.001) and at assessment (42.2 vs. 37.6 yr, p = 0.047) with shorter time between diabetes onset and renal failure (17.8 vs. 22.7 yr, p = 0.3) compared with T1D patients (mean free C-peptide 0.24 ng/mL). In our experience the prevalence of T2D in candidates for SPK is not negligible. Despite some clinical differences with T1D these T2D patients can phenotypically be confounded with T1D in the absence of C-peptide determination.

Pro12Ala polymorphism of the peroxisome proliferator-activated receptor-γ gene in women with polycystic ovary syndrome

Aim. The present study was designed to examine the relationship between Pro12Ala polymorphism of the peroxisome proliferator-activated receptor-γ gene (PPAR-γ) and clinical and hormonal characteristics in women with polycystic ovary syndrome (PCOS).Materials and methods. One hundred patients with PCOS and 100 healthy subjects were included in the study. Serum levels of sex steroids were measured. Insulin resistance was evaluated by homeostasis model assessment (HOMA). The responses of glucose and insulin to an oral glucose tolerance test were analyzed by calculating the respective area under the curve (AUC) by the trapezoidal method. We used the restriction fragment length polymorphism technique and polymerase chain reaction to examine Pro12Ala polymorphism in exon 2 of PPAR-γ.Results. Pro12Ala polymorphism of PPAR-γ was significantly elevated in control subjects (22%) compared with PCOS subjects (15%). All of the Pro12Ala polymorphisms of PPAR-γ were heterozygous. When PCOS subjects with the Pro allele and the Ala allele of PPAR-γ were compared, the latter had lower free testosterone, androstenedione, dehydroepiandrosterone sulfate, insulin and C-peptide levels, as well as lower luteinizing hormone/follicle-stimulating hormone ratio, HOMA insulin resistance index, AUCinsulin, Ferriman–Gallwey score, acne, body mass index and waist-to-hip ratio.Conclusion. We suggest that Pro12Ala polymorphism of the PPAR-γ gene may be a modifier of insulin resistance in women with PCOS.

Gene therapy of streptozotocin-induced diabetes by intramuscular delivery of modified preproinsulin genes.

Despite improvements in insulin preparation and delivery, physiological normoglycemia is not easily achieved in diabetics. Therefore, there has been considerable interest in developing gene therapy approaches to supply insulin. We studied a nonviral muscle-based method of gene therapy and demonstrated that it could prevent hyperglycemia in murine streptozotocin (STZ)-induced diabetes. A plasmid encoding mouse furin-cleavable preproinsulin II cDNA (FI), or its B10-analogue (B10FI), and a plasmid encoding furin were coinjected into muscle of CD-1 mice, who were treated a day later with STZ to induce diabetes. Electroporation was applied to increase gene transfer. Blood glucose was measured in fed and fasting mice, and fasting plasma insulin was measured by radioimmunoassay. The form of insulin produced and the presence of C-peptide were analyzed by gel filtration chromatography. A B10FI plasmid codelivered with a furin plasmid reduced fed and fasting blood glucose levels in STZ-treated diabetic mice. The (pro)insulin levels in plasma were increased by up to 70-fold versus blank plasmid-treated diabetic mice. The administration of FI with furin was less effective. (Pro)insulin levels were greatly increased by using two plasmids carrying different promoter elements (CMV and SV40). Insulin was identified in muscle cells by immunohistochemistry. In plasma, 40-70% of the (pro)insulin was processed to the mature form and free C-peptide was identified. Insulin gene-treated mice had improved growth rates and appeared healthier. A single injection of B10FI with SV40Furin DNA increased plasma (pro)insulin for at least 8 weeks and reduced fed blood glucose levels for 5 weeks and fasting levels for 8 weeks. This is the first report that electroporation-enhanced intramuscular gene therapy with B10FI can prevent hyperglycemia in murine STZ-induced diabetes. Gene therapy using various routes and methods of furin-cleavable insulin gene delivery has been previously explored but, in muscle, results comparable to ours have not been reported.

Hypoglycaemia caused by atypical insulin antibodies in a patient with benign monoclonal gammopathy

We describe a 48-year-old woman with recurrent severe hypoglycaemia apparently caused by a paraprotein with insulin-binding capacity. Very high fasting values were found for serum insulin (170 and > 250 mU l-1) as well as for proinsulin 125 pmol l-1 and an insulinoma was suspected. Hypoglycaemia developed after an oral glucose tolerance (OGTT) test but not during fasting for 48 h. Free insulin and C-peptide were normal during OGTT whereas serum insulin was very high. 125I-insulin binding to serum, determined with a polyethylene glycol (PEG) precipitation method was high (40%), and equally high after addition of 1.7 x 10(-5) mol l-1 cold insulin to estimate non-specific binding. By adding very high concentrations of cold insulin, displacement of 125I-insulin bound to serum was found (50% displacement at 4 x 10(-5) mol l-1). No immunoglobulin G (IgG) insulin antibodies were detected by radioimmunoelectrophoresis. On agarose electrophoresis a small paraprotein (4 g l-1) in the gamma-globulin fraction was detected. 125I-insulin binding to this paraprotein was demonstrated. We conclude that if insulin autoantibodies are suspected as a cause of hypoglycaemia screening for insulin antibodies should always be done with a PEG-precipitation method.

Skeletal muscle beta-adrenoceptor distribution and responses to isoproterenol in hyperthyroidism.

To determine whether hyperthyroidism selectively increases beta-adrenergic receptor density in vessels or fibers of human skeletal muscle, we characterized beta-receptor distribution autoradiographically in muscle biopsies of 18 subjects aged 26 +/- 1 yr before and after daily administration of 100 micrograms 3,5,3'-triiodothyronine (T3) for 2 wk. To establish whether vascular and metabolic responses to beta-adrenergic stimulation are concomitantly altered, we quantified calf blood flow and plasma concentrations of glucose, lactate, glycerol, free fatty acids (FFA), insulin, and C-peptide during graded-dose isoproterenol infusion in eight of these individuals. Differences in beta-adrenergic receptor density among muscle fiber types and vascular components were highly significant (type I greater than type IIa greater than type IIb muscle fibers, P less than 0.001; and type I muscle fibers greater than resistance arterioles, P less than 0.05). Hyperthyroidism increased beta-adrenergic receptor density in all types of muscle fibers (+31-50%; P less than 0.01) but not in resistance arterioles. There was no change in calf blood flow or plasma glucose, glycerol, FFA, insulin, or C-peptide responses to isoproterenol. A rise in lactate during stages 3 and 4 of isoproterenol infusion (P less than 0.01) was observed before but not after T3 administration. Thus hyperthyroidism increases beta-adrenergic receptor density in fibers but not vessels of human skeletal muscle without increasing either metabolic or vascular responses to selective beta-adrenergic stimulation.

A soluble-phase proinsulin radioimmunoassay and its use in diagnosis of hypoglycaemia.

A soluble-phase proinsulin assay has been developed which does not require solid-phase antibody-binding. A human proinsulin standard curve is prepared in insulin-free and proinsulin-free plasma for comparison with unknown plasma samples. Proinsulin and insulin are bound with excess anti-insulin antiserum, and free C-peptide is removed by charcoal adsorption. The supernatant is then assayed using a routine C-peptide radioimmunoassay which utilises anti-C-peptide antiserum. The sensitivity of the assay (2 standard deviations above zero) is 9 pmol/L using 200 microL plasma sample. The assay is free from insulin cross-reactivity up to 100 mU/L and C-peptide up to 2000 pmol/L. Between-assay CV is 13% at 100 pmol/L. The assay has been used in subjects with hypoglycaemia of various aetiologies and has shown that a raised plasma proinsulin in the presence of hypoglycaemia can occur in sulphonylurea-induced and reactive hypoglycaemia as well as in insulinomas. After hyperglycaemic clamps at 7.5, 10 and 15 mmol/L glucose, type II diabetics both on and off sulphonylurea, were found to have lower proinsulin concentrations compared with normal subjects, commensurate with the diabetics' lower insulin responses.

Instability of fasting blood glucose values in noninsulin-dependent diabetic patients with long-term insulin treatment

We studied serum free C-peptide immunoreactivity (CPR) and the coefficient of variation (CV) of fasting blood glucose values (FBG) in 26 insulin-treated patients with non-insulin-dependent diabetes mellitus (NIDDM) in relation to the duration of insulin treatment. Serum free CPR responses during 100 g oral glucose tolerance test (OGTT) were significantly lower in patients with insulin treatment for five years or more than in those with insulin treatment for less than five years although their previous immunoreactive insulin (IRI) responses during OGTT before insulin treatment showed no significant difference. CV of FBG was found to be significantly higher at the time of this study (20.6 ± 7.8%, mean ± SD) than at the second year of insulin treatment (15.3 ± 7.7%, P < 0.05) in the patients with insulin treatment for five or more years but did not show any significant difference in patients with insulin treatment for less than five years at the corresponding times. Thus we measured CV of the FBG in NIDDM patients at various intervals during the long-term insulin or oral hypoglycemic agent treatment in another study. In 20 patients with insulin treatment, CV of FBG was found to be significantly different among the various intervals during insulin treatment ( P < 0.0025). It was significantly higher at the eight year (22.2 ± 8.6%) and 12th year (21.9 ± 9.1%) than at the second year (14.9 ± 6.1%) and fifth year (15.0 ± 6.7%) of insulin treatment ( P < 0.025, P < 0.025; P < 0.05, P < 0.01, respectively). However, in 15 overt NIDDM patients with oral hypoglycemic agent treatment (FBG more than 180 mg/dL before treatment for all). There was no significant difference in the CV of FBG among the various intervals of treatment. The data suggested that the regulatory capacity of FBG in long-term insulin treated NIDDM patients decreases. Although the decrease of endogenous insulin secretion may be a factor, the mechanism needs to be further clarified.

Serum free C-peptide response to oral glucose loading as a parameter for the monitoring of pancreatic B-cell function in diabetic patients

As a parameter for evaluating pancreatic B-cell function, the accuracy of measuring serum free C-peptide immunoreactivity (CPR) was compared with that of measuring plasma immunoreactive insulin (IRI) and urine CPR in diabetic patients during a 100 g oral glucose tolerance test. In 25 non-obese patients receiving oral hypoglycemic agent or diet treatment alone, a positive correlation between the sum of serum free CPR (sigma serum free CPR) and the sum of plasma IRI (sigma plasma IRI) was noted (r = 0.68, P less than 0.001). However, the sum of blood glucose values was found to be negatively correlated to sigma free CPR (r = -0.56, P less than 0.0025), but not to sigma plasma IRI (r = -0.25, NS). In 23 patients receiving diet, oral hypoglycemic agent or insulin treatment, a positive correlation between sigma serum free CPR and urine CPR was noted (r = 0.75, P less than 0.001). However, no significant correlation was found when only insulin-treated patients were investigated (r = 0.37, NS, n = 17). In addition, patients with insulin-dependent diabetes mellitus and non-insulin-dependent diabetes mellitus were better differentiated by measuring sigma serum free CPR than urine CPR. Thus, we concluded that the measurement of serum free CPR during OGTT provides an extremely valuable method for monitoring pancreatic B-cell function in diabetic patients, whether they are receiving insulin treatment or not.

Serum Free C-Peptide Response to Oral Glucose Load in Diabetic Patients

Serum Free C-Peptide Response to Oral Glucose Load in Diabetic Patients

Diagnosis of self-induced hyperinsulinism in an insulin-dependent diabetic patient by radioimmunoassay of free C-peptide.

On the basis of results of simultaneous determinations of plasma free insulin and free c-peptide, episodes of hypoglycemia in an insulin-dependent diabetic were attributed to surreptitious self-administration of insulin. Immunoreactive c-peptide values were falsely increased and diagnostically misleading when measured in unextracted plasma. After preliminary removal of antigen/antibody complexes from the plasma by extraction with polyethylene glycol, the c-peptide values, referred to as "free c-peptide," were suppressed. We suggest that insulin antibodies formed complexes with proinsulin-like material in the plasma of this patient, which accounted for most of the c-peptide immunoreactivity in her unextracted plasma. These complexes must be removed if c-peptide measurements are to be accurate.

Effects of acetylsalicylic acid on plasma glucose, free fatty acid, betahydroxybutyrate, glucagon and C-peptide responses to salbutamol in insulin-dependent diabetic subjects.

The selective beta 2-adrenergic agonist salbutamol increases plasma glucose concentration and the rate of lipolysis when infused in pregnant diabetic women. The aim of the present study was twofold: (a) to focus on the actions of salbutamol on lipid and carbohydrate metabolism in insulin-dependent diabetics: and (b) to investigate possible interferences of acetylsalicylic acid (ASA) with the metabolic responses to i.v. salbutamol. The results obtained during salbutamol infusion (5 microgram/min) in 6 insulin-dependent diabetic subjects demonstrated that this drug caused sustained increases in plasma glucose, free fatty acid and beta-hydroxybutyrate concentrations, as well as a small and transient rise of plasma glucagon. No change in plasma C-peptide concentration occurred during salbutamol. A concurrent infusion of lysine acetylsalicylate reduced the increase in free fatty acids by half, blunted the weak glucagon response but enhanced the rise in plasma glucose following salbutamol administration. The present data show that salbutamol exerts a potent hyperglycemic, lipolytic and ketogenic effect in insulin-dependent diabetics. We suggest that this beta-adrenergic agent should be used cautiously in human diabetes mellitus.

Measurement of serum C-peptide immunoreactivity by radioimmunoassay in insulin-dependent diabetics.

A radioimmunoassay for C-peptide utilizing synthetic C-peptide as an antigen and tyrosylated synthetic C-peptide for iodination was evaluated for its clinical use. Mean fasting C-peptide levels in 24 normal subjects was 2.6 +/- 0.8 ng/ml. During the oral glucose tolerance test, baseline C-peptide in five normal subjects was 1.5 +/- 0.8 ng/ml, and at 60 min was 5.6 +/- 1.6 ng/ml. For two insulin-dependent diabetic patients, diagnoses of factitious hypoglycemia were documented on the basis of simultaneous free insulin and C-peptide determinations. Sera from 24 insulin-dependent diabetics were analyzed for free and total immunoreactive C-peptide and insulin levels. For 20% of juvenile and 64% of maturity-onset diabetics, the presence of proinsulin-like material bound to insulin antibodies was demonstrated by measurement in unextracted serum. This accounted for 20% to 100% of total C-peptide immunoreactivity in these patients. Simple polyethylene glycol precipitation of immune complexes and the measurement of free immunoreactive C-peptide in the supernatant demonstrated subnormal levels (less than 0.5 ng/ml) in all juvenile diabetics and normal levels (1.8 +/- 1.3 ng/ml) in 70% of maturity-onset diabetics.

895 FREE C-PEPTIDE LEVELS IN INFANTS OF DIABETIC MOTHERS: RELATIONSHIP TO HYPOGLYCEMIA, MACROSOMIA, AND HYALINE MEMBRANE DISEASE

Hyperinsulinism is postulated to be related to three major abnormalities in the infant of the diabetic mother (IDM): postnatal hypoglycemia, macrosomia, and hyaline membrane disease (HMD). To test these associations we measured free C-peptide in cord serum of infants of gestational and insulin-requiring diabetics. Free C-peptide levels measure endogenous insulin secretion independent of interfering antibodies. No significant correlation was found between free C-peptide and gestational age in IDM's. IDM's who developed hypoglycemia (serum glucose<30 mg/dl in the first 6 postnatal hours) had significantly (p<0.025) higher cord free C-peptide levels as compared to those without hypoglycemia (7.37±1.23 ng/ml, n=37 vs. 3.91±0.69, n=35, mean±SEM). IDM's with birth weight above the 90th %-ile for gestational age had significantly (p<0.05) higher cord free C-peptide levels as compared to those with birthweight below the 90th %-ile (7.51±1.46, n=31 vs. 4.18±0.60, n=43). 7 IDM's who developed HMD did not have significantly elevated cord free C-peptide levels as compared to 71 IDM's without HMD. The lower than expected incidence of HMD in this series precludes a definitive evaluation of the relationship of hyperinsulinism to HMD in the IDM. We conclude that hyperinsulinism in the IDM, as reflected by free C-peptide level, is directly related to macrosomia and to postnatal hypoglycemia.

Responses of Serum Free C-Peptide Immunoreactivity to Glucose Load in Insulin-Treated Diabetics

Responses of Serum Free C-Peptide Immunoreactivity to Glucose Load in Insulin-Treated Diabetics

Factitious Hypoglycemia

In seven patients with factitious hypoglycemia due to the surreptitious injection of insulin, we made the diagnosis by measurements of plasma insulin and C-peptide immunoreactivity (in seven patients), facilitated by the finding of circulating insulin-binding antibodies (in two patients). The simultaneous demonstration of low plasma glucose, high immunoreactive insulin and suppressed C-peptide immunoreactivity represents a triad of results pathognomonic of exogenous insulin administration. Determination of plasma free C-peptide and free insulin permitted patients with high titers of insulin antibodies, including those with a history of insulin-treated diabetes, to be studied and diagnosed in a way similar to that in subjects who had no circulating insulin antibodies.

Free C-peptide immunoreactivity in insulin-treated diabetics.

The serum samples of healthy and insulin-treated subjects were gel-filtrated before and after acid-alcohol extraction. The sera from insulin-treated diabetics were revealed to contain a variable amount of proinsulin-like components (PLC) which were compounded with insulin antibodies and accumulated in the circulation. These PLC-insulin antibody complexes were found to cross-react with C-peptide antiserum. In this study, we have determined serum C-peptide immunoreactivity (CPR) before and after the removal of PLC-insulin antibody complexes, and expressed as total CPR and free CPR, respectively.The separation of PLC-insulin antibody complexes was performed by the addition of polyethylene glycol to the serum. The free and total CPR values in the subjects never on insulin (7 healthy subjects and 25 adult onset diabetics) were correlated well with each other. The ratios free CPR: total CPR (F/T) were 1.02±0.17 (fasting) and 1.00±0.08 (60min after glucose). In contrast, F/T in insulin-treated diabetics (adult onset 29 cases) were apparently lower than that of the former group ; 0.58± 0.23 (fasting), 0.67±0.22 (60min after glucose). These results provide further evidence that the total CPR values were reflected to a considerable extent by the PLC-insulin antibody complexes. Some cases of insulin-treated diabetics reavealed that over 50% of their serum CPR was attributed to PLC-insulin antibody complexes, and this may be responsible for the higher CPR levels of these cases.Determination of free CPR may provide more accurate index to evaluate the endogenous insulin responses to various stimuli in some of the insulin-treated diabetics.

Control of juvenile diabetes mellitus and its relationship to endogenous insulin secretion as measured by C-peptide immunoreactivity

Proinsulin is converted to insulin and C-peptide in the pancreatic beta-cells; the latter two peptides are secreted in equimolar concentrations. Thus measurements of C-peptide immunoreactivity may provide a means of assessing residual pancreatic function in insulin-treated diabetic patients. Thirty-five patients with a mean (+/- SE) age of 13.4 +/- .6 years who had diabetes mellitus for 4.8 +/- .3 years were included in this study. Glucose and CPR were measured in the fasting state and one hour after 1 gm/kg (maximum 50 gm) of oral and glucose. Patients were assigned to one of two groups on the basis of adequate or poor control of diabetes. Twenty-five of the 35 (71%) patients had evidence of endogenous beta-cell function, i.e., CPR greater than 0.5 ng/ml. CPR levels over 0.5 ng/ml were present in a significantly (p less than 0.05) greater number of patients with diabetes of less than 5 years duration (19/21) than in those with diabetes greater than 5 years duration (6/14). Only one patient showed a rise in CPR after the glucose load. All patients with CPR greater than 2.0 ng/ml were in the adequately controlled groups, but there were patients with CPR less than 2.0 ng/ml in both adequately and poorly controlled groups. Because the CPR value includes both C-peptide and antibody-bound proinsulin, separate determination of free C-peptide was done in 30 patients. These results confirmed the conclusions based on CPR estimation. Although growth hormone values were higher in patients in the poorly controlled group, there was no correlation between hGH and CPR. We conclude that residual insulin secretion in diabetic patients may facilitate good control, but that low CPR values and hence absent beta-cell reserve is not always associated with poor control.

Separation of the two double-chain bovine intermediates of the proinsulin-insulin conversion I. Chemical, immunochemical, circular dichroism, and biological characterization.

The intermediates of the proinsulin-insulin conversion were separated by cation exchange. The circular dichroism spectra of the intermediates showed less alpha-helix than insulin and proinsulin. It is suggested that the C-peptide interacts with the section of alpha-helix contained between residues 2 and 8 in the A-chain of the insulin moieties and unwinds the alpha-helix. The in vivo activities of the intermediates were found to be in the order of 50% relative to insulin. In the fat cell assay, the A-chain-substituted form is weaker (9%) than the B-chain-substituted form (19%). The C-peptide segments of the two forms reacted with C-peptide-specific antibodies as fully as the free C-peptide, on a molar basis. In contrast, the insulin segments were hindered from reacting with insulin-specific antibodies as fully as the insulin.