Islet isolation under cGMP conditions: replacing the coil.

INTRODUCTION: Clinical islet transplantation is increasingly regulated, with isolation standards defined under current good manufacturing practices (cGMPs). cGMP requires validation of equipment cleaning and sterilization. The automated process for islet isolation requires rapid thermal exchange during processing, manipulating a metal coil containing cellular product into and out of chilled/heated waterbaths. We recognize challenges of validating cleaning and sterilization of this coil and propose replacement with a disposable blood warming system. Our specific aims were to assess the system's ability to accommodate flow rates utilized during various phases of pancreatic digestion and to assess its efficiency of heat exchange and temperature control. METHODS: In a pancreas digestion circuit, heat exchange occurred via the coil in a digital water bath, or Warmflo blood warming bag in a digital warming unit. Temperature within the digestion chamber was assessed using an inserted thermocouple. Time to achieve 37 degrees C was measured for set heating element temperatures 38.5 degrees C to 41 degrees C. Circuit temperature maintenance characteristics were also recorded. RESULTS: The Warmflo bloodbag easily accommodated flows of 150 and 300 mL/min. At all set temperatures, the bag resulted in shorter or equivalent time to 37 degrees C than the coil. Maintenance of 37 degrees C was also equivalent. We have utilized this system for four human islet isolations with mean time to 37 degrees C of 5.5 minutes, without difference in digestion quality. CONCLUSIONS: Use of a disposable blood warming system in place of the coil during islet isolation provides adequate flow characteristics, heat exchange, and temperature control and may facilitate evolution of islet isolation toward cGMP compliance.

Calpains play a role in insulin secretion and action.

Studies of the genetic basis of type 2 diabetes suggest that variation in the calpain-10 gene affects susceptibility to this common disorder, raising the possibility that calpain-sensitive pathways may play a role in regulating insulin secretion and/or action. Calpains are ubiquitously expressed cysteine proteases that are thought to regulate a variety of normal cellular functions. Here, we report that short-term (4-h) exposure to the cell-permeable calpain inhibitors calpain inhibitor II and E-64-d increases the insulin secretory response to glucose in mouse pancreatic islets. This dose-dependent effect is observed at glucose concentrations above 8 mmol/l. This effect was also seen with other calpain inhibitors with different mechanisms of action but not with cathepsin inhibitors or other protease inhibitors. Enhancement of insulin secretion with short-term exposure to calpain inhibitors is not mediated by increased responses in intracellular Ca2+ or increased glucose metabolism in islets but by accelerated exocytosis of insulin granules. In muscle strips and adipocytes, exposure to both calpain inhibitor II and E-64-d reduced insulin-mediated glucose transport. Incorporation of glucose into glycogen in muscle also was reduced. These results are consistent with a role for calpains in the regulation of insulin secretion and insulin action.

Overexpression of Bcl-x(L) in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion.

To study effects of Bcl-x(L) in the pancreatic beta-cell, two transgenic lines were produced using different forms of the rat insulin promoter. Bcl-x(L) expression in beta-cells was increased 2- to 3-fold in founder (Fd) 1 and over 10-fold in Fd 2 compared with littermate controls. After exposure to thapsigargin (10 microM for 48 h), losses of cell viability in islets of Fd 1 and Fd 2 Bcl-x(L) transgenic mice were significantly lower than in islets of wild-type mice. Unexpectedly, severe glucose intolerance was observed in Fd 2 but not Fd 1 Bcl-x(L) mice. Pancreatic insulin content and islet morphology were not different from control in either transgenic line. However, Fd 2 Bcl-x(L) islets had impaired insulin secretory and intracellular free Ca(2+) ([Ca(2+)](i)) responses to glucose and KCl. Furthermore, insulin and [Ca(2+)](i) responses to pyruvate methyl ester (PME) were similarly reduced as glucose in Fd 2 Bcl-x(L) islets. Consistent with a mitochondrial defect, glucose oxidation, but not glycolysis, was significantly lower in Fd 2 Bcl-x(L) islets than in wild-type islets. Glucose-, PME-, and alpha-ketoisocaproate-induced hyperpolarization of mitochondrial membrane potential, NAD(P)H, and ATP production were also significantly reduced in Fd 2 Bcl-x(L) islets. Thus, although Bcl-x(L) promotes beta-cell survival, high levels of expression of Bcl-x(L) result in reduced glucose-induced insulin secretion and hyperglycemia due to a defect in mitochondrial nutrient metabolism and signaling for insulin secretion.

Adaptation to hyperglycemia enhances insulin secretion in glucokinase mutant mice.

The present study was undertaken to test the hypothesis that exposure to high glucose concentrations enhances insulin secretion in pancreatic islets from glucokinase-deficient mice. Insulin secretion and intracellular calcium ([Ca2+]i) were measured as the glucose concentration was increased from 2 to 26 mmol/l in islets from heterozygous glucokinase (GK)-deficient mice (GK+/-) and their wild-type littermates (GK+/+). Results obtained in islets incubated in 11.6 or 30 mmol/l glucose for 48-96 h were compared. GK+/- islets that had been incubated in 30 mmol/l glucose showed improved although not normal insulin secretory and [Ca2+]i responses to the standard glucose challenge as well as an enhanced ability to sense small amplitude glucose oscillations. These effects were associated with increased glucokinase activity and protein. In contrast, exposure of GK+/+ islets to 30 mmol/l glucose increased their basal insulin secretion but reduced their incremental secretory responses to glucose and their ability to detect small amplitude glucose oscillations. Thus exposure of GK+/- islets to 30 mmol/l glucose for 48-96 h enhanced their ability to sense and respond to a glucose stimulus, whereas similar exposure of GK+/+ islets induced evidence of beta-cell dysfunction. These findings provide a mechanistic framework for understanding why glucokinase diabetes results in mild hyperglycemia that tends not to increase over time. In addition, the absence of one allele of the glucokinase gene appears to protect against glucose-induced beta-cell dysfunction (glucose toxicity).

Defective pancreatic beta-cell glycolytic signaling in hepatocyte nuclear factor-1alpha-deficient mice.

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene cause maturity onset diabetes of the young type 3, a form of type 2 diabetes mellitus. In mice lacking the HNF-1alpha gene, insulin secretion and intracellular calcium ([Ca2+]i) responses were impaired following stimulation with nutrient secretagogues such as glucose and glyceraldehyde but normal with non-nutrient stimuli such as potassium chloride. Patch clamp recordings revealed ATP-sensitive K+ currents (KATP) in beta-cells that were insensitive to suppression by glucose but normally sensitive to ATP. Exposure to mitochondrial substrates suppressed KATP, elevated [Ca2+]i, and corrected the insulin secretion defect. NAD(P)H responses to glucose were substantially reduced, and inhibitors of glycolytic NADH generation reproduced the mutant phenotype in normal islets. Flux of glucose through glycolysis in islets from mutant mice was reduced, as a result of which ATP generation in response to glucose was impaired. We conclude that hepatocyte nuclear factor-1alpha diabetes results from defective beta-cell glycolytic signaling, which is potentially correctable using substrates that bypass the defect.

Apoptosis in insulin-secreting cells. Evidence for the role of intracellular Ca2+ stores and arachidonic acid metabolism.

This study investigated the role of intracellular free Ca2+ concentration ([Ca2+]i) in apoptosis in MIN6 cells, an insulin secreting cell line, and in mouse islets. Thapsigargin, an inhibitor of sarcoendoplasmic reticulum Ca2+-ATPases (SERCA), caused a time- and concentration-dependent decrease in the viability of MIN6 cells and an increase in DNA fragmentation and nuclear chromatin staining changes characteristic of apoptosis. Two structurally distinct SERCA inhibitors, cyclopiazonic acid and 2,5-di-[t-butyl]-1,4-hydroquinone also caused apoptosis, but agents that increased [Ca2+]i by other mechanisms did not induce apoptosis in MIN6 cells. Carbachol- or ionomycin-releasible intracellular Ca2+ stores were completely depleted in cells treated by SERCA inhibitors, but not by other agents that increase [Ca2+]i. The ability of thapsigargin to induce cell death was not affected by blocking Ca2+ influx or by clamping [Ca2+]i with a cytosolic Ca2+ buffer suggesting that the process did not depend on changes in [Ca2+]i per se. However, application of the lipoxygenase inhibitors 5,8,11-eicosatrienoic acid and nordihydroguaiaretic acid partially prevented MIN6 cell apoptosis, while exposure of cells to the product of lipoxygenase, 12-hydroxy-[5,8,10,14]-eicosatetraenoic acid, caused apoptosis. In contrast, inhibition of cyclooxygenase with indomethacin did not abolish thapsigargin-induced apoptosis in MIN6 cells. Our findings indicate that thapsigargin causes apoptosis in MIN6 cells by depleting intracellular Ca2+ stores and leading to release of intermediate metabolites of arachidonic acid metabolism.

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.

Changes in pancreatic islet glucokinase and hexokinase activities with increasing age, obesity, and the onset of diabetes.

We examined changes in high- and low-Km glucose phosphorylating activity in pancreatic islet extracts from the prediabetic Zucker diabetic fatty (ZDF) rat between 5-6 weeks and 12 weeks of age (after the onset of diabetes). Comparisons were made between the activity observed in the ZDF rat and that seen in the ZDF lean control (ZLC) rat and the obese nondiabetic Zucker fatty (ZF) rat. At 5-6 weeks of age, insulin resistant ZDF and ZF rats were hyperinsulinemic, compared with the ZLC rat, but had normal plasma glucose levels. Kinetic parameters (Vmax and Km for glucose) of hexokinase (HK) and Km of glucokinase (GCK) did not differ between groups. Islet GCK activity for ZDF and ZF rats was 1.7-fold greater than in ZLC rats (P < 0.02 and P < 0.001, respectively). By 12 weeks of age, hypersecretion of insulin at 5.0 mmol/l glucose was observed in perifused islets from both obese groups relative to the ZLC rat. Islets from ZDF rats failed to increase insulin secretion in response to increased glucose concentration. Group differences in the kinetic parameters for GCK or in the Km values for HK were not significant. Islet HK activity for ZDF and ZF rats was 1.9-fold (P < 0.05) and 1.7-fold (P < 0.05) greater, respectively, than for ZLC rats. Compared with the 5- to 6-week-old animals, HK activity increased 3.1-fold (P < 0.001), 2.5-fold (P < 0.002), and 2.0-fold (P < 0.05) for ZDF, ZF, and ZLC rats, respectively. Differences in GCK activity between 5- to 6- and 12-week-old rats were not significant for any of the groups. We conclude: 1) increased islet glucose phosphorylating activity is present in insulin resistant and hyperinsulinemic ZF and ZDF rats, relative to the ZLC rat; 2) at 12 weeks of age, hyperinsulinemic ZDF and ZF rats demonstrated significant increases in HK activity, compared with lean controls; and 3) deficiency in GCK activity does not explain failure of diabetic ZDF islets to respond to glucose, since differences between diabetic ZDF and nondiabetic ZF rats were not statistically significant. Increases in pancreatic islet phosphorylating activity seem to be important in maintaining basal hyperinsulinemia in insulin-resistant animals, but do not appear to play a role in the progression to glucose intolerance and diabetes.

A novel point mutation in the insulin gene giving rise to hyperproinsulinemia.

A 58-yr-old obese white Caucasian male type 2 diabetic, entered into the UK Prospective Diabetes Study, was found to have raised fasting total proinsulin levels 708 pmol/L(-1) (normal range, 3-16 pmol/L(-1)) and normal specific plasma insulin level 29 pmol/L(-1) (normal range, 21-75 pmol/L(-1)). Immunoreactive plasma insulin, measured by RIA, was 503 pmol/L(-1). DNA was extracted, the insulin gene amplified by the PCR, and by direct sequencing, a novel point mutation, G1552C, was identified, which resulted in the substitution of proline (CCT) for arginine (CGT) at position 65. This prevented cleavage of the C-peptide A-chain dibasic cleavage site (lys-arg) by the processing protease in the pancreatic beta-cells. The plasma proinsulin and insulin levels were in accord with expression of both the wild-type and the mutant alleles. The G1552C mutation was not linked with diabetes, because it was present in a 37-yr-old nondiabetic daughter and not in a 35-yr-old daughter who had had gestational diabetes.

Measurement of proinsulin and intermediates. Validation of immunoassay methods by high-performance liquid chromatography.

Human proinsulin and 32-33 split proinsulin have been measured in the peripheral circulation by immunoradiometric assays (IRMAs) and have been shown to be elevated in impaired glucose tolerance and non-insulin-dependent diabetes mellitus (NIDDM). The IRMA for 32-33 split proinsulin did not discriminate between this molecule and des-32 or des-31,32 split proinsulin. We describe the comparison of IRMA for human plasma proinsulin and 32-33 split proinsulins with assays combined with high-performance liquid chromatography (HPLC), which can discriminate between 32-33 split, des-32 split, and des-31,32 split proinsulin. Subjects were those with normal glucose tolerance (n = 8) and those with NIDDM (n = 17), who were studied while fasting and 30 min after a glucose load. After collection, blood was centrifuged promptly, and the serum/plasma was stored frozen until assay. Both IRMA and HPLC methods were calibrated against synthetic peptides. Interassay coefficients of variation for the IRMA for proinsulin and 32-33 split proinsulin were < 13% over the ranges 3.8-65 pmol/l and 6.4-65 pmol/l, respectively. The following regression lines were obtained: proinsulin IRMA = -0.143 + 1.066 HPLC, r = 0.860; 32-33 split proinsulin IRMA = 0.048 + 1.051 HPLC; and des-31,32 split proinsulin, r = 0.814. For both analytes, there was no significant difference in the relationship of IRMA to HPLC results between the various subject groups and various time points. Thus, the IRMA for proinsulin has been validated by an independent method.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in the patterns of insulin secretion before and after diagnosis of IDDM.

OBJECTIVE: To study the natural history of beta-cell dysfunction in an individual who developed insulin-dependent diabetes mellitus (IDDM) over a 13-month period while under observation. RESEARCH DESIGN AND METHODS: Insulin secretion rates (ISR) in response to intravenous glucose and mixed meals were estimated by deconvolution of C-peptide levels. RESULTS: When fasting glucose and glycosylated hemoglobin concentrations were still within the normal range, insulin secretory responses to intravenous glucose infusion were reduced, but 80- to 100-min secretory oscillations could still be detected. Sequential glucose infusion studies over a 3-month period demonstrated a progressive reduction in insulin secretion. The tight temporal coupling between ultradian oscillations in ISR and glucose observed in nondiabetic subjects was lost. In response to mixed meals, the oscillatory pattern of secretion was preserved, but the magnitude of the secretory responses was reduced. CONCLUSIONS: Our results indicate that despite the lower absolute secretory rates, ultradian ISR oscillations persist in the period before and immediately after the onset of IDDM in this subject, but they are less tightly coupled to glucose than in nondiabetic subjects.

Alterations in pulsatile insulin secretion in the Zucker diabetic fatty rat.

Insulin secretion from the isolated perfused pancreas is characterized by pulses occurring every 5-15 min. The present experiments were performed to explore the role of glucose in regulating these pulses. The pancreata from 12 Wistar (W), 12 Zucker diabetic fatty (ZDF), and 6 nondiabetic lean Zucker control (ZC) male rats were isolated and perfused at 37 degrees C with an oxygenated Krebs Ringer solution containing bovine serum albumin and glucose. In W and ZDF, insulin secretion was pulsatile during constant glucose, as assessed by pulse analysis (ULTRA). The pulse period in W was significantly shorter than in ZDF (7.1 +/- 0.6 vs. 14.7 +/- 1.0 min; P < 0.001), whereas the median relative pulse amplitude was not different. When glucose was administered as a series of 10-min sine waves, spectral analysis showed that the normalized spectral power at 10 min was greater in W and ZC compared with ZDF (34.2 +/- 5.9 and 32.9 +/- 2.9 vs. 3.2 +/- 0.9; P < 0.0001), demonstrating entrainment of the insulin pulses to the exogenous glucose oscillations in W and ZC but not in ZDF. Furthermore, in ZDF, the insulin secretory rates were not higher when 28 mM rather than 7 mM glucose were used. In additional studies, islets of Langerhans from one W, three ZDF, and three ZC rats were isolated and perifused using an oscillatory glucose concentration. Single and groups of islets were studied. Islets from diabetic rats demonstrated the same lack of entrainment by glucose seen in the perfused pancreas, suggesting that the defect is at the cellular level.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in immunoreactive proinsulin and insulin clearance induced by weight loss in NIDDM.

Subjects with overt non-insulin-dependent diabetes mellitus (NIDDM) were studied in comparison to obese nondiabetic control subjects and patients with subclinical diabetes. Pancreatic insulin secretion rates were measured by deconvolution of peripheral C-peptide over a 24-h period while subjects consumed an isocaloric mixed diet. Subjects were then placed on caloric restriction for at least 6 weeks, during which time body weight fell by at least 10%. Refeeding with solid mixed meals was then resumed for at least 2 weeks until isocaloric intake was attained, and then the meal profiles were repeated. Before weight loss, insulin, C-peptide, and insulin secretion rates were significantly higher in subjects with subclinical diabetes than in the other two groups. Proinsulin concentrations were significantly greater in the two diabetic groups than in control subjects, but, when expressed as a percentage of the total insulin immunoreactivity, the differences were significant only in the group with overt diabetes. Weight loss because of hypocaloric feeding resulted in a significant increase in the rate of clearance of endogenously secreted insulin but did not affect the clearance of C-peptide. In obese subjects and those with subclinical diabetes, weight loss was associated with a reduction in insulin secretion rates, presumably as a result of improvements in insulin sensitivity. In patients with overt diabetes and hyperglycemia, weight loss improved beta-cell responsiveness to glucose and was associated with an increase in insulin clearance and a reduction in proinsulin immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma insulin, C-peptide, and proinsulin concentrations in obese and nonobese individuals with varying degrees of glucose tolerance.

Conventional immunoassays to quantify insulin concentration do not differentiate between insulin and proinsulin. Thus, previous conclusions as to the relationship between the development of hyperglycemia in patients with noninsulin-dependent diabetes mellitus (NIDDM) and pancreatic insulin secretory function may have been confounded by not being able to determine the contribution made by plasma proinsulin to the putative measurements of plasma insulin concentration in these patients. The current study was initiated to address this issue by making specific measurements of plasma insulin, proinsulin, and C-peptide concentrations in 42 individuals: 14 with normal glucose tolerance, 12 with impaired glucose tolerance (IGT), and 16 with NIDDM. The study population was further subdivided into a nonobese (body mass index, < 30 kg/m2) and an obese (body mass index, > 30 kg/m2) group. Mixed meals were given at 0800, 1200, and 1800 h, and blood was removed at 0800 h (before the meal) and at hourly intervals from then until 1600 h. Plasma glucose concentrations throughout the sampling period were slightly, but significantly (P < 0.01), greater in patients with IGT than in normal individuals. Patients with NIDDM had markedly elevated glycemic excursions, greater than either of the other two groups (P < 0.002). Both plasma immunoreactive insulin and C-peptide concentrations from 0800-1600 h were higher (P < 0.002-0.001) in patients with either IGT or NIDDM than in the group with normal glucose tolerance. Although day-long plasma immunoreactive insulin and C-peptide concentrations were higher, on the average, in patients with IGT compared to those with NIDDM, the difference was not statistically significant. Plasma proinsulin concentrations were highest in patients with NIDDM (P < 0.002), lower in those with normal glucose tolerance (P < 0.002), and intermediate in patients with IGT. When the calculated "true" insulin concentration was determined by taking the proinsulin content into consideration, patients with IGT had the highest day-long levels, with the lowest values found in the control population (P < 0.002). Although absolute values varied as a function of obesity, the generalizations outlined above were found in both weight groups. These results show that ambient plasma proinsulin concentrations increase as glucose tolerance declines. However, true plasma insulin concentrations in response to mixed meals remain highest in patients with IGT, lowest in normal individuals, and intermediate in patients with NIDDM. Thus, previous conclusions that absolute day-long plasma insulin concentrations are not lower than normal in patients with NIDDM do not appear to result from an inability to differentiate true insulin from proinsulin.

Hypoglycemia due to surreptitious injection of insulin. Identification of insulin species by high-performance liquid chromatography.

OBJECTIVE: To identify the circulating species of insulin after separation by high-performance liquid chromatography (HPLC) in patients with factitious hypoglycemia. RESEARCH DESIGN AND METHODS: In three of four patients presented, the diagnosis of surreptitious insulin injection was made by documenting the presence of animal insulin in the circulation after separation of the circulating insulin forms by HPLC. RESULTS: Animal insulin was identified. CONCLUSIONS: Thus, the identification of the circulating form of insulin in the circulation by HPLC may be a useful adjunct in the diagnosis of factitious hypoglycemia if animal insulin has been injected and if the simultaneously measured concentrations of insulin and C-peptide are inconclusive.

Lack of effect of high-dose biosynthetic human C-peptide on pancreatic hormone release in normal subjects.

We studied the effect of high doses of biosynthetic human C-peptide on pancreatic hormone secretion in response to oral (75 g) and intravenous [( IV] 0.33 g/kg of D50%) glucose on normal volunteers. The infusion of human C-peptide at a rate of 360 ng/kg/min body weight, increased the plasma C-peptide concentration from a basal level of 0.32 +/- 0.04 pmol/mL to 38.5 +/- 1.8 pmol/ml. Overall, C-peptide had no significant effect on the serum levels of glucose, insulin, proinsulin, glucagon, and pancreatic polypeptide, either under basal conditions or following IV and oral glucose administration. However, small decreases in glucose and insulin concentrations that were not statistically significant were seen during the first hour after C-peptide infusion. The results of the present studies are therefore consistent with the conclusion that even supraphysiologic plasma concentrations of infused C-peptide do not affect basal insulin secretion or overall insulin secretory responses to oral or IV glucose. However, we cannot definitively exclude a small reduction in insulin secretion in the first hour after oral glucose ingestion.

Ingestion of a mixed meal does not affect the metabolic clearance rate of biosynthetic human C-peptide.

The validity of C-peptide as a peripheral marker of insulin secretion during different physiological conditions depends on the demonstration that C-peptide clearance is constant under these circumstances. Recently biosynthetic human C-peptide, identical in structure to pancreatic human C-peptide, became available for use in human subjects. The present study was undertaken to determine if the metabolic clearance of C-peptide was altered by ingestion of a mixed meal. Eight insulin-dependent diabetic patients received constant iv infusions of biosynthetic human C-peptide which raised the plasma C-peptide concentration to a level of 3.8 +/- 0.2 (+/- SEM) pmol/ml. The MCR of C-peptide was 4.5 +/- 0.3 ml/kg X min. After steady state levels of C-peptide had been reached, each patient consumed a 530 calorie mixed meal. The plasma glucose concentration increased from a baseline value of 104.5 +/- 4.8 mg/dl to a 336 +/- 10 mg/dl 150 min later. This change in plasma glucose was not associated with a significant alteration in the plasma C-peptide concentration and the MCR of the infused C-peptide was not affected by meal ingestion (4.5 +/- 0.3 vs. 4.3 +/- 0.3 ml/kg X min). These results therefore support the validity of using C-peptide as a marker for changes in insulin secretion after mixed meals.