Meal-induced 24-hour profile of circulating glycated insulin in type 2 diabetic subjects measured by a novel radioimmunoassay

Increasing evidence supports a role for glycated insulin in the insulin-resistant state of type 2 diabetes. We measured 24-hour profiles of plasma glycated insulin, using a novel radioimmunoassay (RIA), to evaluate the effects of meal stimulation and intermittent fasting on circulating concentrations of plasma glycated insulin in type 2 diabetes. Patients (n = 6; hemoglobin A(1c) [HbA(1c)], 7.2% +/- 0.6%; fasting plasma glucose, 7.4 +/- 0.7 mmol/L; body mass index [BMI], 35.7 +/- 3.5 kg/m(2); age, 56.3 +/- 4.4 years) were admitted for 24 hours and received a standardized meal regimen. Half-hourly venous samples were taken for plasma glycated insulin, glucose, insulin, and C-peptide concentrations between 8 am and midnight and 2-hourly overnight. The mean plasma glycated insulin concentration over 24 hours was 27.8 +/- 1.2 pmol/L with a mean ratio of insulin:glycated insulin of 11:1. Circulating glucose, insulin, C-peptide, and glycated insulin followed a basal and meal-related pattern with most prominent increments following breakfast, lunch, and evening meal, respectively. The mean concentrations of glycated insulin during the morning, afternoon, evening, and night-time periods were 24.4 +/- 2.5, 28.7 +/- 2.3, 31.1 +/- 2.1, and 26.2 +/- 1.5 pmol/L, respectively, giving significantly higher molar ratios of insulin:glycated insulin of 18.0:1, 14.2:1, and 12.7:1 compared with 7.0:1 at night (P <.01 to P <.001). These data demonstrate that glycated insulin circulates at relatively high concentrations in type 2 diabetes with a diurnal pattern of basal and meal-stimulated release. A higher proportion of glycated insulin circulates at night suggestive of differences in metabolic clearance compared with native insulin.     

Sample preparation and radioimmunoassay for circulating free and antibody-bound insulin concentrations in insulin-treated diabetics: a re-evaluation of methods

Methods for blood sample preparation and radioimmunoassay of free and antibody-bound serum or plasma insulin concentrations, using polyethylene glycol (PEG) solution to precipitate bound insulin, have been evaluated and compared. Method A was rapid mixing of whole blood with PEG, followed by separation of bound insulin by centrifugation into Ficoll; method B was rapid PEG addition to whole blood, followed by centrifugation alone; method C was conventional PEG addition to thawed plasma or serum, followed by centrifugation to remove bound insulin. Method A was found to have acceptable performance and reproducibility; serum free insulin levels were not significantly different from those measured by the simpler method B. In samples from insulin-dependent diabetics, serum free insulin was significantly higher and bound insulin significantly lower in method A compared to the conventional method C. However, in samples from non-diabetics there was no significant difference between methods A and C. Spurious results were obtained by addition of PEG and assay of plasma and serum samples after either freezing and thawing or incubation at 37 degrees C for 2h, compared to PEG addition and assay of fresh plasma or serum samples. We conclude that conventional, delayed addition of PEG to plasma or serum may result in redistribution of free and bound insulin and that values more representative of in vivo blood insulin levels are obtained by rapid PEG addition methods.     

Intramuscular injection of insulin lispro or soluble human insulin: pharmacokinetics and glucodynamics in Type 2 diabetes.

AIM: The aim of the study was to compare the pharmacokinetics and glucodynamics of insulin lispro and soluble human insulin following intramuscular (i.m.) injection in patients with Type 2 diabetes with secondary failure of sulphonylureas. METHODS: Single 15-U i.m. doses of insulin lispro or soluble human insulin were administered to 16 patients in a two-way, randomized, crossover design. Glucodynamic and pharmacokinetic parameters were determined over 6 h after insulin injection using clamp techniques. RESULTS: Insulin C(max) was significantly higher (971 +/- 217 vs. 659 +/- 141 pmol/l, P < 0.001) and T(max) was significantly shorter (46.9 +/- 27 vs. 94.7 +/- 50.1 min, P = 0.002) with insulin lispro. Glucose infusion rate (GIR) curves showed clear separation 20 min after injection and were significantly greater for insulin lispro during the 40-60, 60-80 and 80-100-minute time intervals. Total glucose infused was only approximately 5% larger with insulin lispro during the 6-h follow-up, due to lower insulinaemia at later time points. The glucose R(max) and TR(max) were not statistically different between insulin treatments. CONCLUSION: This study shows that i.m. injection of insulin lispro is followed by its more rapid absorption, which results in stronger metabolic effect in the first 2 h when compared with soluble human insulin under the same test conditions. Diabet. Med. 18, 562-566 (2001)     

Fasting and post-prandial glycemia and their correlation with glycated hemoglobin in Type 2 diabetes

OBJECTIVE: The relative contribution of fasting and post-prandial glucose to glycated hemoglobin (HbA1c) is controversial. In the present study, we assessed the relationship with HbA1c of fasting and post-prandial glucose measured in a more naturalistic setting, through home glucose self-monitoring or with a continuous glucose monitoring system (CGM). MATERIALS AND METHODS: A consecutive series of 300 patients with Type 2 diabetes were enrolled in the study, provided that they performed blood glucose self-monitoring. HbA1c and fasting plasma glucose (FPG) were measured at enrolment. RESULTS: Both fasting plasma and capillary glucose showed a significant correlation with HbA1c (r=0.66 and 0.61, respectively; p<0.001). When home glucose monitoring was considered, both mean fasting and post-prandial glucose showed a significant correlation with HbA1c (r=0.71 and 0.73, respectively). In patients in the lower tertile of body mass index (BMI), HbA1c showed a significant correlation at multivariate analysis with post-prandial glucose, but not with fasting glucose. In patients with HbA1c >7%, both fasting and post-prandial glucose showed a significant correlation, after adjustment for age and BMI, with HbA1c (both p<0.01); conversely, in those with HbA1c < or =7%, such a correlation could be observed for fasting (p<0.01), but not for post-prandial glucose. CONCLUSION: In conclusion, both fasting and post-prandial glucose contribute to the determination of HbA1c . Home glucose self-monitoring appears to provide a more accurate assessment of metabolic control than a single plasma glucose measurement in experimental conditions. Fasting glucose could provide a greater contribution to HbA1c in patients with lower HbA1c, while post-prandial glucose seems to play a major role in leaner Type 2 diabetic subjects.     

Irregular circulating insulin concentrations in type 2 diabetes mellitus: an inverse relationship between circulating free fatty acid and the disorderliness of an insulin time series in diabetic and healthy individuals

Insulin is released in a high-frequency pulsatile secretory pattern, which is reflected as quantifiable oscillations in peripheral circulating insulin concentrations. Type 2 diabetes mellitus is characterized by a broad spectrum of abnormalities in beta-cell function, including disturbed pulsatile insulin secretion as assessed by autocorrelation analysis. To achieve further insight into beta-cell pathophysiology in type 2 diabetes, we examined the orderliness of the baseline serum insulin time series (blood collection every minute for 75 minutes) in 16 type 2 diabetics (fasting plasma glucose, 170 +/- 10 mg/dL [mean +/- SE]; serum free fatty acid [FFA], 0.794 +/- 0.083 mmol/L; and known diabetes duration, 6 +/- 2 years) and 15 healthy controls (serum FFA, 0.523 +/- 0.055 mmol/L). We used approximate entropy (ApEn), a recently introduced scale- and model-independent measure of serial irregularity. ApEn was significantly increased in the type 2 diabetics compared with the controls (0.671 +/- 0.016 v 0.653 +/- 0.008, P = .04), indicating more irregular serum insulin time series in diabetics. Autocorrelation also discriminated between groups, although only when the data were pooled. Interestingly, an inverse relationship between ApEn and serum FFA was observed in the controls (r = -.63, P = .01) and diabetics (r = -.65, P < .01), whereas no relationships were found between ApEn and the age, body mass index (BMI), or plasma glucose. In conclusion, type 2 diabetes is characterized by an increased disorderliness of the fasting serum insulin time series, strongly suggesting perturbed rapid oscillatory insulin release. An inverse relationship between ApEn and fasting serum FFA among both groups might suggest a hitherto unknown stabilizing action of FFA on the high-frequency pulsatile insulin release process. This hypothesis needs to be tested in experimental designs that more specifically focus on this issue, eg, during changes in serum FFA.     

Effects of nateglinide on the secretion of glycated insulin and glucose tolerance in type 2 diabetes

AIMS: Glycation of insulin has been demonstrated within pancreatic beta-cells and the resulting impaired bioactivity may contribute to insulin resistance in diabetes. We used a novel radioimmunoassay to evaluate the effect of nateglinide on plasma concentrations of glycated insulin and glucose tolerance in type 2 diabetes. METHODS: Ten patients (5 M/5 F, age 57.8+/-1.9 years, HbA(1c) 7.6+/-0.5%, fasting plasma glucose 9.4+/-1.2 mmol/l, creatinine 81.6+/-4.5 microM/l) received oral nateglinide 120 mg or placebo, 10 min prior to 75 g oral glucose in a random, single blind, crossover design, 1 week apart. Blood samples were taken for glycated insulin, glucose, insulin and C-peptide over 225 min. RESULTS: Plasma glucose and glycated insulin responses were reduced by 9% (P=0.005) and 38% (P=0.047), respectively, following nateglinide compared with placebo. Corresponding AUC measures for insulin and C-peptide were enhanced by 36% (P=0.005) and 25% (P=0.007) by nateglinide. CONCLUSIONS: Glycated insulin in type 2 diabetes is reduced in response to the insulin secretagogue nateglinide, resulting in preferential release of native insulin. Since glycated insulin exhibits impaired biological activity, reduced glycated insulin release may contribute to the antihyperglycaemic action of nateglinide.     

Nephropathy in Type 1 diabetes is associated with increased circulating activated platelets and platelet hyperreactivity

Patients with diabetes mellitus (DM) have increased platelet activation compared to non-diabetic controls. Platelet hyperreactivity has been associated with adverse cardiovascular outcomes in Type 2 DM, and with diabetic nephropathy. We investigated the relationship between platelet activation and nephropathy in Type 1 DM. Patients with Type 1 DM and diabetic nephropathy (n = 35), age- and sex-matched Type 1 DM patients with persistent normoalbuminuria (n = 51), and healthy age- and sex-matched controls (n = 30) were studied. Platelet surface P-selectin, platelet surface activated GPIIb/IIIa, monocyte-platelet aggregates (MPAs) and neutrophil-platelet aggregates (NPAs) were measured by whole blood flow cytometry as markers of platelet activation. Platelet reactivity was assessed in response to exogenously added ADP and thrombin receptor activating peptide (TRAP). Platelet surface P-selectin (basal and in response to 0.5 or 20 µM ADP) was higher in nephropathy patients compared with normoalbuminuric patients (P = 0.027), and non-diabetic controls (P = 0.0057). NPAs were higher in nephropathy patients compared to normoalbuminuric patients (P = 0.0088). MPAs were higher in nephropathy patients compared to non-diabetic controls (P = 0.0075). There were no differences between groups in activated GPIIb/IIIa or in response to TRAP at any end-point. More patients with nephropathy received aspirin (71.4%) compared to normoalbuminuric patients (27.4%) (P < 0.0001). Type 1 diabetic nephropathy, as compared with normoalbuminuria, is associated with circulating activated platelets and platelet hyperreactivity to ADP, despite the confounding variable of more nephropathy patients receiving aspirin. This platelet activation is likely to contribute to the known increased risk of cardiovascular events in patients with diabetic nephropathy.     

Demonstration of a novel circulating anti-prostacyclin receptor antibody

Although coronary artery disease (CAD) is appreciated to be accelerated in patients with chronic spinal cord injury (SCI), the underlying mechanism of CAD in SCI remains obscure. We have recently shown that platelets from subjects with SCI develop resistance to the inhibitory effect of prostacyclin (PGI₂) on the platelet stimulation of thrombin generation. The loss of the inhibitory effect was due to the loss of high-affinity prostanoid receptors, which may contribute to atherogenesis in SCI. Incubation of normal, non-SCI platelets in SCI plasma (n = 12) also resulted in the loss of high-affinity binding of PGI₂ (Kd₁ = 9.1 ± 2.0 nM; n₁ = 170 ± 32 sites per cell vs. Kd₁ = 7.2 ± 1.1 nM; n₁ = 23 ± 8 sites per cell), with no significant change in the low-affinity receptors (Kd₂ = 1.9 ± 0.1 μM; n₂ = 1,832 ± 232 sites per cell vs. Kd₂ = 1.6 ± 0.1 μM; n₂ = 1,740 ± 161 sites per cell) as determined by Scatchard analysis of the binding of [³H]PGE₁. The loss of high-affinity PGI₂ binding led to the failure of PGI₂ to inhibit the platelet-stimulated thrombin generation. The increase of cellular cyclic AMP level, mediated through the binding of PGI₂ to low-affinity receptors in platelets, was unaffected in SCI platelets. PAGE and immunoblot of SCI plasma showed the presence of an IgG band, which specifically blocked the binding of [³H]PGE₁ to the high-affinity PGI₂ receptors of normal platelets. PAGE of the reduced IgG band, the amino acid sequence of the novel band as a heavy chain of IgG that inhibits the binding of [³H]PGE₁ to the high-affinity platelet PGI₂ receptor, demonstrates that the specific recognition and inhibition of high-affinity PGI₂ binding to platelets was due to an anti-prostacyclin receptor antibody present in SCI plasma.     

Increased plasma glycated low-density lipoprotein concentrations in diabetes: a marker of atherogenic risk

Nonenzymatic glycation of apolipoprotein B in the low-density lipoprotein (LDL) complex has been considered a proatherogenic modification contributory to the increased susceptibility of patients with diabetes to atherosclerosis. We postulated that glycated LDL concentrations might be associated with other markers of cardiovascular disease. To explore this hypothesis, we measured glycated LDL concentrations by a monospecific immunoassay in 50 patients with type 1 and 100 patients with type 2 diabetes and examined relationships with the amount of albumin excretion and the serum cholesterol and triglyercide concentrations. Plasma glycated LDL showed a significant positive correlation (r = 0.325; P < 0.001) with urinary albumin excretion that was higher in type 1 (r = 0.463) than in type 2 (r = 0.245) patients. The mean glycated LDL concentration progressively increased with increasing albumin excretion when patients were subcategorized into groups of normoalbuminuria, low (相似文献   

Development of a specific radioimmunoassay to measure physiological changes of circulating leptin in cattle and sheep

Studies of leptin in large domestic ruminants have been limited to measurements of gene expression because methods to measure circulating levels are not available. To develop a bovine leptin radioimmunoassay, we produced recombinant bovine leptin and used it to immunize rabbits, and to prepare bovine leptin tracer and standards. A single antiserum with sufficient affinity and titer was identified. Using this antiserum, logit-transformed binding of (125)I-labeled bovine leptin was linearly related (R(2)= 0.99) to the log of added bovine or ovine leptin between 0.1 to 2.0 ng. Serial dilution of bovine and ovine plasma, chicken serum and bovine milk gave displacement curves that were parallel to those of bovine or ovine leptin. Recoveries of external addition of bovine leptin in ewe and cow plasma ranged between 94 and 104%. Plasma leptin concentration measured by this assay was directly related to the plane of! nutrition in growing calves and lambs. At 11-14 weeks of age, ewe lambs had a higher circulating leptin concentration than ram lambs. Finally, plasma leptin concentration was linearly related to the fat content of the empty carcass in growing cattle and to body condition score in lactating dairy cows. We conclude that circulating leptin in sheep and cattle is increased by fatness and plane of nutrition, consistent with results in humans and rodents. This assay provides an important tool to investigate mechanisms that regulate plasma leptin in cattle and sheep.     

Free and total insulin integrated concentrations in insulin dependent diabetes

Studies of the 24 hr insulin concentration profiles in diabetic subjects on chronic exogenous insulin have been hampered by the presence of endogenous anti-insulin antibody, which gives spurious estimates of radioimmunoassayable insulin concentrations. The introduction of polyethylene glycol precipitation of endogenous antibody has allowed development of reliable assays for determiniation of free and total insulin concentration in subjects on insulin therapy. This article reports our observations of plasma free and total insulin concentration in 50 Type I and Type II ambulatory insulin dependent diabetics, utilizing a continuous 24 hr blood withdrawal technique. In response to exogenous insulin, study subjects had marked elevations in insulin concentrations compared to controls. Mean free insulin integrated concentration was 3.5-fold higher in diabetics than nondiabetics. Mean total insulin integrated concentration was 868 μU/ml, more than 20 times in excess of total insulin concentration in nondiabetics. There was a wide range among diabetics in the percentage of total insulin in the free insulin fraction. Neither free nor total insulin integrated concentration correlated with dose of exogenous insulin. Free and total insulin concentration profiles showed a limited range of variation in insulin concentration during the 24 hr of study, no subject having a profile that mimicked that observed in nondiabetic subjects. Glucose integrated concentration showed no correlation with free insulin integrated concentration, however, it did correlate inversely with the percentage of total insulin in the free insulin fraction. These data emphasize the difficulty in establishing normal patterns of insulin among diabetic subjects on conventional subcutaneous insulin therapy.     

Mathematical modelling of changes in circulating insulin and C-peptide concentrations

Summary A simple mathematical model is proposed to assess the validity of methods currently used in clinical investigation to detect short-term or long-term alterations in insulin and/or C-peptide clearance. This model draws attention to unwarranted conclusions which resulted from the analysis of insulin and C-peptide plasma concentrations.     

Intracellular hyperinsulinism: a metabolic characteristic of obesity with and without Type 2 diabetes: intracellular insulin in obesity and Type 2 diabetes

There is evidence that intracellular insulin may carry out some insulin mediated actions, including glucose transport. As intracellular insulin has never been quantitatively assessed in human cells, we evaluated its concentrations in monocytes from normal subjects (n = 7) and obese patients without (n = 9) and with Type 2 diabetes mellitus (n = 10). After the incubation of cells with labeled insulin for 60 min at 37 degrees C, intracellular intact insulin concentrations were measured by HPLC and expressed as pmol x 10(-6). Insulin concentrations were higher (ANOVA P < 0.01) within cells from obese (115.4 +/- 26.4 pmol x 10(-6)/2 x 10(5) cells) and obese diabetic patients (93.2 +/- 36.3 pmol x 10(-6)/2 x 10(5) cells) compared with normal cells (28.5 +/- 13.1 pmol x 10(-6)/2 x 10(5) cells). Moreover, after insulin was removed from the incubation medium the decrease of intracellular insulin was significantly lower (P < 0.01) in cells from both obese and obese diabetic patients than in normal subjects. Intracellular undissociated insulin-insulin receptor complexes on average, increased 2-fold (P < 0.01) in cells from insulin resistant patients compared with normal cells. Finally, in downregulated cells from obese and obese diabetic patients, the recycling of the internalized insulin receptor was completely disrupted. In conclusion, monocytes from obese patients with and without Type 2 diabetes mellitus, present increased intracellular insulin concentrations and these conditions are associated with a significant impairment of insulin receptor processing. Increased intracellular insulin concentration in cells from these patients may be necessary in order to overcome insulin resistance.