Prolonged exposure of human beta-cells to high glucose increases their release of proinsulin during acute stimulation with glucose or arginine

The disproportionate hyperproinsulinemia in type 2 diabetes has been attributed to either a primary beta-cell defect or a secondary dysregulation of beta cells under sustained hyperglycemia. This study examines the effect of a 10- to 13-day exposure to 20 mmol/L glucose on subsequent proinsulin and insulin release by human islets isolated from nondiabetic donors. Compared to control preparations kept at 6 mmol/L glucose, the high glucose cultured beta-cells released more proinsulin and less insulin during perifusion at 5, 10, or 20 mmol/L glucose. The lower amounts of secreted insulin resulted from a marked reduction in cellular insulin content (5-fold lower than in controls). The higher amount of secreted proinsulin is attributed to the sustained state of cellular activation that is known to occur after prolonged exposure to high glucose levels. This activated state of the beta-cell population is also held responsible for its higher secretory responsiveness to 5 mmol/L arginine at a submaximal (5 mmol/L) glucose concentration (8-fold higher proinsulin levels than in the control population). It results, together with the reduction in cellular insulin content, in 7- to 10-fold higher proinsulin over insulin ratios in the medium; at 5 mmol/L glucose, this extracellular ratio is similar to that in the cells. These data add direct support to the view that a disproportionate hyperproinsulinemia can result from a sustained activation of human beta-cells after prolonged exposure to elevated glucose levels.     

Effects of human placental lactogen and growth hormone on the production of insulin and somatomedin C/insulin-like growth factor I by human fetal pancreas in tissue culture

We have investigated the ability of glucose, human GH and human placental lactogen (hPL) to alter the content and release of somatomedin C/insulin-like growth factor I (SM-C/IGF-I), and the biosynthesis, content and release of insulin from cultured human fetal pancreas. Fetal pancreatic explants obtained from glands following prostaglandin-induced abortion between 12 and 21 weeks of gestation were maintained in free-floating culture for 3-5 days before the experiments. The explants were then cultured for 3 days in medium containing either 2.7 or 16.7 mmol glucose/l with or without GH (4.5 or 45.5 nmol/l) or hPL (4.6 or 46.5 nmol/l). Serum-free medium from the final 24 h of culture was collected and SM-C/IGF-I and insulin were measured radioimmunologically in both conditioned medium and tissue explants extracted with acid ethanol. Insulin biosynthesis, determined by immunoprecipitation of [3H]leucine incorporated into insulin, was not significantly altered by any experimental variable. Incubation in the presence of 16.7 mmol glucose/l caused an increase of insulin release from explants, but had no consistent action on insulin content, compared with medium containing 2.7 mmol glucose/l. The pancreatic content and release of SM-C/IGF-I were independent of these glucose concentrations. Neither GH nor hPL altered insulin or SM-C/IGF-I content or release in the presence of the lower glucose concentration. At the higher glucose concentration, 45.5 nmol GH/l did not alter insulin release but caused a significant increase in SM-C/IGF-I content.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of human proinsulin on glucose turnover and intermediary metabolism

We compared the effects of human proinsulin and human insulin on glucose disposal, suppression of hepatic glucose production (HGP), and intermediary carbohydrate and lipid metabolism. Six young, lean, subjects underwent eight separate euglycemic clamps with low-dose intravenous (IV) infusions of insulin and proinsulin (four each). The insulin infusions gave steady-state levels of 0.08 +/- 0.004 (I1), 0.12 +/- 0.003 (I2), 0.18 +/- 0.07 (I3), and 0.25 +/- 0.06 nmol/L (I4). The proinsulin infusions were chosen to give steady-state levels approximately 20-fold higher on a molar basis than insulin, based on previous findings that proinsulin has only 5% to 10% the biological potency of insulin. Steady-state proinsulin levels were 1.2 +/- 0.04 (P1), 2.8 +/- 0.07 (P2), 4.5 +/- 0.3 (P3), and 6.9 +/- 0.3 nmol/L (P4). HGP was suppressed equally by proinsulin and insulin at the four dose levels. Percentage elevation of glucose disposal was significantly increased during each of the insulin infusions compared with proinsulin: I1 107% +/- 4%, P1 87% +/- 4% (P = .03); I2 143% +/- 7%, P2 125% +/- 12% (P = .01); I3 238% +/- 38%, P3 173% +/- 22% (P = .03); I4 283% +/- 17%, P4 178% +/- 11% (P = .002). Dose-response curve analysis demonstrated that proinsulin stimulated glucose disposal approximately 3.3% compared with insulin. The effectiveness of proinsulin in suppressing HGP was approximately 5% compared with insulin. Plasma nonesterified fatty acids, blood glycerol, and 3-hydroxybutyrate were suppressed by similar amounts during each of the four insulin and proinsulin doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activators of protein kinase C stimulate insulin secretion from the human fetal pancreas

The human fetal pancreas, like the adult pancreas, releases insulin in response to agents that increase intracellular levels of calcium and cyclic AMP. It differs from the adult pancreas in that glucose causes either minimal or no insulin secretion from it. Whether activation of protein kinase C will cause release of insulin from this immature cell, as it does from the adult pancreas, is unknown. This was the phenomenon examined in the experiments below. Activators of protein kinase C, 1.3 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) and 25.1 microM 1-oleoyl-2-acetylglycerol, caused significant release of insulin from cultured explants of human fetal pancreas. Lower concentrations of TPA--0.65 microM or less--had no such effect. Agents that are known to inhibit protein kinase C, 100 microM 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, 2 mM polymyxin B, and 1 mM dibucaine, caused abolition of the insulinogenic effect of 1.3 microM TPA. Glucose, at 2.8, 5, and 10 mM, also inhibited the positive effect of TPA. However, when the concentration of glucose was increased to 20 or 30 mM, stimulation of insulin secretion returned to levels achieved with TPA alone. These levels were significantly higher than the minimal response achieved with 20 mM glucose. Such data support the involvement of protein kinase C activation in insulin secretion from the human fetal pancreas. It is postulated that this involvement may occur physiologically.     

Relative hyperproinsulinemia as a sign of islet dysfunction in women with impaired glucose tolerance.

Proinsulin release is increased relative to insulin secretion in subjects with type 2 diabetes, indicative of islet dysfunction. However, it has not been conclusively shown whether there is an increased relative proinsulin release in subjects with impaired glucose tolerance (IGT), i.e. whether it precedes the development of diabetes. We therefore determined the proinsulin to insulin ratios in the fasting state and after acute stimulation of insulin secretion in 23 postmenopausal women, aged 61-62 yr (mean +/- SD, 61.7 +/- 0.5 yr). Ten women had normal glucose tolerance (NGT), and 13 had IGT. The groups were matched for insulin sensitivity and did not differ in body mass index. Proinsulin and insulin secretion were measured after arginine stimulation (5 g, i.v.) at three glucose levels (fasting, 14 mmol/L, and >25 mmol/L), and the acute insulin (AIR(arg)) and proinsulin responses (APIR(arg)) were calculated as the mean 2-5 min postload increase. At fasting glucose, levels of insulin, proinsulin, or the proinsulin/insulin ratio (13.6 +/- 5.0% vs. 11.1 +/- 2.7%; P = NS) did not differ between NGT and IGT. Although the AIR(arg) values were decreased in the IGT group at all glucose levels (P < 0.05), the absolute proinsulin levels and the APIRs(arg) were similar between IGT and NGT women. Therefore, the IGT women had higher proinsulin/insulin ratios at 14 mmol/L (10.7 +/- 4.4% vs. 6.4 +/- 1.8%; P = 0.006) and more than 25 mmol/L glucose (11.4 +/- 5.2% vs. 6.7 +/- 2.1%; P = 0.007). The IGT group had increased APIR(arg)/AIR(arg) at fasting (2.2 +/- 1.4% vs. 1.3 +/- 0.6%; P = 0.047) and more than 25 mmol/L glucose (3.5 +/- 1.6% vs. 2.3 +/- 0.7%; P = 0.037). We conclude that women with IGT exhibit increased relative proinsulin secretion, suggesting a defect in the intracellular proinsulin processing before diabetes develops.     

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

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

Organ culture of human foetal pancreas: conditions which affect basal and stimulated insulin release

Human foetal pancreas has been maintained in organ culture with net synthesis and release of insulin for up to 60 days. The age of the donor foetus affected the basal insulin release rate. A plateau of secretion was reached with foetuses of greater than or equal to 16 weeks of gestation. Explants cultured within 2 h of expulsion following prostaglandin induced termination secreted 3.0 times more insulin after 20 days of culture than those cultured within 2-4 h and 8.1 times more than those cultured more than 4 h post-termination. A high oxygen environment was toxic to the explants during culture. Fresh tissue responded to a high concentration of glucose (19.3 mM) with a small but significant increase in insulin secretion. The addition of 10 mM theophylline caused a major increase in insulin release. Cultured tissue did not respond to glucose alone but did not show increased insulin release following stimulation with glucose (22 mM) together with theophylline (10 mM) in static incubation. The culture of human foetal pancreatic tissue may be useful in maintaining responsive beta cells and may help to ensure an adequate amount of donor tissue for future transplantation into diabetic patients.     

Insulin release from a cloned precursor beta cell line

This paper describes the establishment in long-term tissue culture of a functional, clonal beta (B) cell line UMR 407/3 derived from neonatal rat pancreas. Immunofluorescence demonstrated specific and uniform staining for insulin. Transmission electron microscopy showed the presence of microvilli and cytoplasmic granules. The doubling time in culture was approximately 60 h in 2% (v/v) fetal calf serum with inhibition of growth at confluence. Biochemical studies demonstrated the incorporation of [3H]leucine into proinsulin and insulin, with insulin comprising 43.6% of the total radioactivity incorporated into immune complexes. When incubated at 37 degrees C for 30 min with Krebs-Ringer bicarbonate buffer (pH 7.4), the amount of insulin released on stimulation by 16.7 mmol glucose/l, 20 mmol DL-glyceraldehyde/l or 20 mmol alpha-ketoisocaproate/l was significantly higher compared with 5.6 mmol glucose/l. The mean insulin content was equivalent to 99 +/- 0.4 fmol (S.E.M.)/5 X 10(5) cells. Regulated insulin release was maintained through at least 15 passages in culture. The cells showed morphological evidence of senescence after passage 26 and this was associated with significant reduction in stimulated insulin release as well as insulin content. The ability of the cells of this clonal line to grow in soft agar suggests that it is a precursor cell line. The clonal B cell lines isolated so far may thus represent variably committed rather than fully differentiated B cells in culture. These clonal non-neoplastic cell lines will be useful models with which to study the regulation of maturation/differentiation of B cells and insulin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolonged maximal stimulation of insulin secretion in healthy subjects does not provoke preferential release of proinsulin.

Release of immature secretory granules rich in incompletely processed proinsulin has been proposed to explain the relative hyperproinsulinemia in type 2 diabetic and insulinoma patients because of a constant secretory drive resulting from hyperglycemia and autonomous secretion, respectively. To test this hypothesis, insulin secretion was stimulated by a combination of hyperglycemia (11 mmol/L clamp), intravenous (i.v.) tolbutamide (1 g), and i.v. glucagon (initial bolus 10 micrograms/kg body weight, maintenance infusion 2 micrograms/kg body weight per hour) for 3 h. Circulating IR-insulin and IR-C-peptide concentrations increased 89-fold and 14-fold over basal values, respectively, but IR-proinsulin concentrations increased only ninefold over basal values. Estimation of the amount of insulin secreted (based on deconvolution analysis of plasma C-peptide values) showed that approximately 76 +/- 21 U were secreted during the stimulation period. This amount is a significant proportion of pancreatic insulin content in normal humans. In molar terms, IR-proinsulin (integrated incremental response multiplied by metabolic clearance rate of proinsulin) relative to IR-C-peptide (= insulin) secretion (deconvolution analysis) was estimated to be equal or even lower than the known proportion in islets (0.22 +/- 0.05%). Thus, using a near-maximal stimulation of insulin secretion maintained long enough to cause release of amounts of insulin approaching the estimated pancreatic content, no preferential release of proinsulin was observed in normal humans. Therefore, the hyperproinsulinemia of type 2 diabetes and in insulinoma patients may be caused by additional defects in the proinsulin to insulin conversion process.     

A DIRECT ASSAY FOR PROINSULIN IN PLASMA AND ITS APPLICATIONS IN HYPOGLYCAEMIA

A direct radioimmunoassay in unextracted plasma is described. The assay has a sensitivity of 4 pmol/l (2 standard deviation from zero). The proinsulin antiserum was immuno-adsorbed against human C-peptide and insulin coupled to glass beads. Cross-reactivity of the antiserum was assessed and shown to be less than 0.01% with both peptides. In normal healthy fasting subjects the plasma proinsulin level was 6.7 +/- 1.7 pmol/l (n = 17) (mean +/- SD). Fasting proinsulin levels in non-insulin dependent diabetics were significantly elevated compared with non diabetics (14.2 +/- 2 pmol/l (n = 11) vs 6.7 +/- 1.7 (n = 17) P less than 0.005). The insulin/proinsulin ratio was 3.4:1 in the non-insulin dependent diabetic compared with 6:1 in non-diabetics. Samples from 21 insulinoma patients were assayed and mean fasting plasma proinsulin level was 255 pmol/l +/- 479 when the patients were hypoglycaemic. The range in pro-insulin levels was large (30-2300 pmol/l). Mean fasting proinsulin level in three hypoglycaemic subjects due to sulphonylurea overdose was 15.7 +/- 2.3 pmol/l. The molar ratio of proinsulin to insulin was 1:6 in healthy subjects, 1:1 in insulinoma patients and 10:1 in sulphonylurea induced hypoglycaemic patients.     

Growth hormone and metasomatotrophic diabetes: effects on insulin and proinsulin of serum and pancreas in dogs

Summary In normal fasting dog serum, the insulin: proinsulin molar proportion was 71:29%. In response to glucose infusion, the proinsulin proportion decreased. In the pancreas, the proinsulin proportion was lower than in serum. Growth hormone treatment for one day increased serum insulin sevenfold and proinsulin 18-fold. The proinsulin proportion increased to 49%. The growth hormone injections magnified the response to glucose infusion. The rise in serum insulin was 16 times the normal, proinsulin also rose but its proportion decreased. Growth hormone treatment for 6 days decreased pancreatic insulin to 5% and proinsulin to 46% of normal. In the permanent (metasomatotrophic) diabetes produced by the prolonged administration of growth hormone, serum insulin decreased and the proinsulin proportion increased. No rises in serum insulin nor proinsulin occurred following glucose infusion. In the pancreas, insulin and proinsulin were reduced to 1.6% and 8% of normal. The reduction in the immunoreactive insulin of the pancreas was more pronounced in the tail than in the head and body regions. The results indicate that in the state of augmented insulin secretion and hyperinsulinaemia produced by growth hormone and in the reduced insulin secretion and hypoinsulinaemia of metasomatotrophic diabetes, the proportion of proinsulin in serum is increased due to beta cell secretion containing a higher proportion of proinsulin than normal.     

Morphology, yield and functional integrity of islet-like cell clusters in tissue culture of human fetal pancreata obtained after different means of abortion

Morphology, yield and function were studied in cultured islet-like cell clusters (ICC) from 140 human fetal pancreata obtained after abortions of different types performed at 11-23 weeks of gestation (12 by hysterotomy, 75 by mechanical dilation and extraction, and 53 induced with prostaglandin). After collagenase digestion and culture in medium supplemented with 10% human serum, up to 2000 free-floating ICC were formed from a single pancreas. Randomly scattered insulin- and glucagon-immunoreactive cells were found in the medullary part of the ICC. More than 100 ICC developed in 100% of the hysterotomies and 87% of the mechanical abortions, but in only 53% of the prostaglandin-induced abortions. Insulin and glucagon levels in the culture medium decreased rapidly during the first 7 days of culture, but then remained stable for at least 31 days. The hysterotomy-derived ICC responded to 10 mmol/l theophylline plus 20 mmol/l glucose by a 12.2 +/- 3.1 (SEM, N = 7) fold increase in insulin release, as compared with a 5.4 +/- 0.9 fold response of the prostaglandin ICC (N = 16; P less than 0.02). Despite the low proportion of B-cells, (pro)insulin biosynthesis accounted for 10% of the total protein biosynthesis in low (2 mmol/l) glucose. In conclusion, the yield and viability of the ICC were clearly better, if prostaglandin had not been used for the induction of the abortion.     

Production of insulin-like growth factor-II by human fetal pancreas in culture

Insulin-like growth factor-II (IGF-II) is a polypeptide hormone thought to be involved in fetal development because of its wide distribution in fetal tissues and its presence in the fetal circulation. We have developed a highly sensitive radioreceptor assay for IGF-II using rat liver microsomal membranes and have used this assay to test for the presence of IGF-II in the human fetal pancreas and the release of IGF-II by the human fetal pancreas in organ culture. IGF-II was present in extracts of pancreatic tissue (0.056 +/- 0.012 pmol/mg tissue, n = 5) and was released in culture at the rate of 0.027-0.134 pmol/mg tissue per day with release being maintained for at least 3 weeks in culture. The rate of release was not affected by the gestational age of the fetus over 14 to 20 weeks but was significantly related to the rate of insulin release (r = 0.712, P less than 0.001, n = 34). Chronic exposure to 12-0-tetradecanoylphorbol-13-acetate (TPA), which inhibits insulin release in the human fetal pancreas, caused an 85% drop in IGF-II production, which was reversed when TPA was removed. These studies demonstrate that IGF-II is produced by the human fetal pancreas in a pattern similar to that of insulin. We suggest that control of IGF-II release, like that of insulin, may involve protein-kinase C and that IGF-II may have a paracrine or autocrine role in the development of fetal pancreatic function.     

Indirect two-site immunoradiometric assay of rat and mouse proinsulin

Summary An indirect two-site immunoradiometric assay for rat and mouse proinsulin using a rabbit antibody to synthetic rat C-peptide has been developed. The sensitivity of the assay is 0.006 pmol/ml. Proinsulin was 4.95% of the total proinsulin and insulin in extracts of rat pancreas and 5.45% in extracts of isolated rat islets. The mean fasting rat insulin and proinsulin concentrations were 0.13±0.09 pmol/ml (n=5) and 0.008±0.002 pmol/ml (n=5) respectively. The mean fasting mouse proinsulin concentration was 0.019±0.006 pmol/ml (n=8). In rats intravenous glucose produced a biphasic insulin response but proinsulin rose progressively to 0.021±0.011 pmol/ml at 45 min. In mouse oral glucose increased the proinsulin concentration to 0.13 pmol/ ml at 30 min. Proinsulin release from isolated rat islets was studied during intermittent or continuous high glucose (20 mmol/l) stimulation in static incubation. Significant increases in proinsulin release were only observed 90 min after initial exposure to high glucose whether glucose stimulation was continuous or intermittent. Both in vivo and in vitro glucose stimulation led initially to a fall in the proinsulin/ insulin molar ratio but later upon prolonged stimulation this progessively increased to above the basal value.     

A fifteen-month double-blind cross-over study of the efficacy and antigenicity of human and pork insulins

The antigenicity and efficacy of semi-synthetic human and pancreatic pork insulins have been compared in a double-blind double cross-over study in 96 insulin-treated diabetic patients. Transfer from pork to human insulin was associated with a 1.2 +/- 0.5 mmol/l deterioration (p less than 0.05) in the fasting blood glucose level, while the opposite change caused a 1.1 +/- 0.5 mmol/l improvement (p less than 0.05). After 4 months treatment, glycosylated haemoglobin levels were lower on pork (11.1 +/- 0.3%) than on human (11.7 +/- 0.3% p less than 0.01) insulin. The incidence of hypoglycaemia was similar with the two insulins. IgG insulin antibody levels were identical after human insulin treatment (5.7 +/- 0.4 micrograms/l) compared to pork insulin treatment (5.9 +/- 0.5 micrograms/l). Patients with high levels of antibodies (greater than 10 micrograms/l) showed a similar reduction in level when switched to either species of highly purified insulin. The deterioration in fasting blood glucose control is consistent with similar reports for biosynthetic human insulin and suggests, in the absence of changes in insulin antibody levels, a small but clinically significant pharmacokinetic difference between human and pork insulin.     

Insulin and glucagon secretory responses to arginine, glucagon, and theophylline during perifusion of human fetal islet-like cell clusters

The secretory responsiveness of human fetal pancreatic endocrine cells was studied by perifusion of cultured islet-like cell clusters (ICC). ICC were obtained from 7 fetuses at 13-15 weeks gestation and 21 fetuses at 17-22 weeks gestation. The ICC were challenged with glucose (20 mmol/L), arginine (10 mmol/L), glucagon (1.4 mumol/L), and theophylline (10 mmol/L) combined with zero, low (2 mmol/L), or high (20 mmol/L) glucose. At 13-15 weeks, glucose and arginine enhanced insulin release in some experiments, whereas glucagon and theophylline were always potent stimuli (mean response, 4-fold regardless of the glucose concentration). At 17-22 weeks, both glucose alone (20 mmol/L) and arginine (10 mmol/L, with 2 mmol/L glucose) induced a small (1.4- to 1.5-fold) increase in insulin release. When arginine was combined with 20 mmol/L glucose, the response was potentiated to become a 2.3-fold increase. In contrast, glucagon was equally effective in 2 and 20 mmol/L glucose (2.9- and 2.6-fold responses, respectively) and produced a half-maximal response even in the absence of glucose. In this age range the most potent stimulus for insulin release was clearly theophylline. The effect of theophylline was also remarkably independent of the glucose concentration of the perifusate (5.6-, 8.1-, and 8.6-fold responses at 0, 2, and 20 mmol/L glucose, respectively). Glucagon release from the ICC of the 17- to 22-week-old fetuses was low (mean basal glucagon release, 2.9; insulin, 24.8 fmol/100 ICC/min). Glucagon release was not affected by 20 mmol/L glucose, but was stimulated by arginine and theophylline. These findings suggest that in the fetal pancreas, in contrast to the adult organ, insulin release results from elevation of intracellular cAMP concentrations (by glucagon or theophylline) relatively independent of the exogenous glucose concentration. Therefore, glucagon may have an important role in regulating insulin release during the early development of human fetal B-cells.     

Evidence against a rate-limiting role of proinsulin processing for maximal insulin secretion in subjects with impaired glucose tolerance and beta-cell dysfunction

In subjects with impaired glucose tolerance (IGT) insulin secretion is impaired. Increased proinsulin/insulin (PI/I) ratios suggest that there is also reduced processing of proinsulin to insulin in this condition. The PI/I ratio in the insulin secretory granule is ideally assessed by plasma measurements in response to acute stimulation of insulin secretion. In the present study we tested the hypothesis that maximal stimulation of insulin secretion results in exhaustion of the proinsulin conversion pathway to insulin. We therefore determined the PI/I ratio in 11 normal glucose-tolerant subjects (NGT) and 11 subjects with IGT in response to glucose (squarewave hyperglycemic clamp, 10 mmol/L), glucagon-like peptide-1 (GLP-1; primed-continuous infusion), and arginine given during the continued GLP-1 infusion. In IGT, insulin levels were significantly lower during the first phase (144 +/- 20 vs. 397 +/- 119 pmol/L; P = 0.02), at the end of the GLP infusion (2142 +/- 350 vs. 5430 +/- 1091 pmol/L; P: = 0.002), and in response to arginine (3983 +/- 375 vs. 8663 +/- 1430 pmol/L; P = 0.005). In response to glucose, the minimum PI/I ratio was significantly higher in IGT (3.4 +/- 0.6%) than in NGT (1.4 +/- 0.5%; P = 0.02), suggesting defective proinsulin processing in this condition. In subjects with IGT, the PI/I ratio decreased significantly after GLP-1 priming (1.7 +/- 0.2%; P = 0.02) and after arginine given during GLP-1 (1.4 +/- 0.2%; P = 0.007) and was not significantly different from those values in NGT (1.3 +/- 0.2% and 1.3 +/- 0.2%, respectively; both P = NS). In conclusion, during maximal stimulation of insulin secretion in subjects with IGT, the PI/I ratio in plasma decreased significantly and was not different from that in normal controls. This strongly argues against the hypothesis that defective processing of proinsulin to insulin represents a major component of the beta-cell dysfunction in IGT.     

Tissue culture of human fetal pancreas: growth hormone stimulates the formation and insulin production of islet-like cell clusters

The human fetal pancreas (HFP) is a potential source of insulin-producing B-cells for transplantation to insulin-dependent diabetic patients. We recently described a technique for culturing HFP tissue in vitro which results in the development of islet-like cell clusters (ICC). These clusters exhibited (pro)insulin biosynthesis and a modest rate of insulin secretion, and immunocytochemical staining indicated the presence of insulin-positive cells in the cell clusters. In this study this technique was used to evaluate the effects of the addition of 1000 micrograms/L GH to HFP cultured in medium RPMI-1640 plus 10% human serum. ICCs developed in 21 of 33 consecutive cultures. GH increased the yield of ICC by 35% compared to explants supplemented with human serum alone. The insulin content of the ICCs also was increased, but the size of individual ICCs was not affected by GH, as reflected by an unchanged DNA content. GH also caused increased insulin release when the ICCs were stimulated with 16.7 mM glucose plus 5 mM theophylline. However, (pro)insulin biosynthesis was not affected by the addition of GH. These results suggest that GH stimulates the formation of both ICCs and insulin production within the explants. These observations are relevant both for the production of human fetal B-cells intended for transplantation into insulin-dependent diabetic patients and for our knowledge of the growth regulation of the HFP B-cell.     

Development of insulin and proinsulin secretion in newborn pony foals

At birth, the endocrine pancreas must assume a glucoregulatory role if the neonate is to survive the transition from parenteral to enteral nutrition. In species like the horse, neonatal hypoglycaemia is common, which suggests that the glucoregulatory mechanisms are not always fully competent at birth. Hence, this study examined pancreatic beta cell function in newborn foals during nutritional adaptation over the first 10 days post partum. Over a 48 h period at three time intervals after birth (days 1-2, 5-6 and 9-10 post partum), the beta cell responses to suckling and to intravenous administration of glucose, arginine and saline were measured in seven normal pony foals. Basal plasma concentrations of proinsulin, but not insulin or glucose, increased significantly between days 1 and 10. Suckling caused a gradual increase in plasma glucose, which was accompanied by a significant increase in plasma insulin concentrations 15 min after the onset of suckling on days 5 and 9, but not day 1. There was no significant change in plasma proinsulin concentrations in response to suckling at any age. At all ages studied, glucose and arginine administration stimulated an increase in the plasma concentrations of insulin and proinsulin; these beta cell responses did not change significantly with postnatal age. The insulin responses to glucose were significantly greater than those of arginine at each time period. Glucose clearance was significantly slower on day 1 than subsequently. Proinsulin and glucose, but not insulin, concentrations decreased significantly after saline administration at all three ages. At each time period, there was a significant positive relationship between the plasma insulin and proinsulin concentrations, the slope of which was significantly shallower on days 1-2 than subsequently. These results show that equine beta cells are responsive to glucose and arginine and release both insulin and proinsulin during the immediate postnatal period. They also suggest that newborn foals may be insulin resistant on the first day after birth.     

The effects of human proinsulin on glucose turnover and intermediary metabolism in insulin-dependent-diabetes mellitus.

We have compared the action of human proinsulin and insulin on glucose turnover, intermediary carbohydrate, and lipid metabolism in insulin-dependent-diabetic (IDDM) subjects. Six, young, weight-matched (23 +/- 2 kg-2) IDDM subjects underwent separate hyperinsulinemic euglycemic clamps. Three, low dose, iv infusions of both insulin and proinsulin were used to construct dose response curves. The proinsulin infusions were chosen to give steady state levels approximately or equal to 20-fold higher on a molar basis than insulin, based on previous findings that proinsulin has only 5-10% the biological potency of insulin. Hepatic glucose production, measured using [6'6'2H2]glucose, was suppressed equally by proinsulin and insulin at the three dose levels; (I1) 2.8 +/- 0.7 (P1) 3.3 +/- 0.6, (I2) 2.3 +/- 0.9 (P2) 3.3 +/- 1.1, (I3) -2.0 +/- 1.7 (P3) -1.1 +/- 0.6 mumol/kg min-1. Percentage elevation of glucose disposal was significantly increased during the insulin infusions compared to proinsulin; (I1) 132 +/- 12 (P1) 78 +/- 4 p < 0.01; (I2) 157 +/- 18 (P2) 104 +/- 14; P < 0.05; (I3) 242 +/- 23 (P3) 159 +/- 24 p = 0.02. Dose response curve analysis demonstrated that proinsulin stimulated glucose disposal approximately or equal to 3.7% whereas suppression of HGP was congruent to 5.7% compared to insulin. Proinsulin had a significantly weaker effect than insulin, at the lowest infusion dose, in percent suppression of plasma nonesterified fatty acids (I1 34 +/- 4, P1 14 +/- 15%; P < 0.05), blood glycerol (I1 47 +/- 4, P1 30 +/- 3%; P < 0.01) and 3-hydroxybutyrate levels (I1 81 +/- 7, P1 42 +/- 17%; P < 0.05). Proinsulin caused significant net reductions in blood lactate levels compared to insulin at each infusion dose; (P1) -130 +/- 34, (I1) -32 +/- 30 mumol/L (P < 0.05) (P2) -139 +/- 76 (I2) +8 +/- 65 mumol/L (P < 0.05) (P3) 48 +/- 60 (I3) 230 +/- 64 mumol/L (P < 0.05). We conclude that in IDDM: 1) proinsulin has a preferential effect on the liver compared to muscle, in terms of glucose handling; 2) proinsulin may have a different effect on lactate metabolism compared to insulin; 3) proinsulin at the lowest dose resulted in an inability to suppress lipolysis and ketogenesis; 4) glucose turnover can be underestimated using [6'6'2H2]glucose.