(Pro-)Insulin Biosynthesis and Release of Newly Synthesized (Pro-)Insulin from Isolated Islets of Rat Pancreas in the Presence of Amino Acids and Sulphonylureas+,++

Abstract. The influence of arginine, lysine, tolbutamide and glibenclamide on (pro-)insulin biosynthesis and release of newly synthesized (pro-)insulin was studied in isolated islets of rat pancreas. Islets were incubated with ³H-leucine and glucose in the presence and absence of the test agents. Proinsulin and insulin of islets and incubation media were separated by gel filtration on Sephadex G 50. Estimations were carried out for radioactivity and immunoreactivity for insulin. All four test substances were able to enhance insulin release whereas no stimulation of leucine incorporation into (pro-)insulin was found. Arginine and tolbutamide even markedly reduced (pro-)insulin synthesis. Conversion of proinsulin to insulin was not affected by any of the test agents. For studying the influence of the 4 substances on secretion of newly synthesized (pro-)insulin two experimental models were used: 1) Labelling of the islets in the presence of the test agents, followed by uniform stimulation with glucose alone in the presence or absence of Ca⁺⁺. 2) Addition of the 4 test substances after uniform prelabelling of the islets. 1) Presence of arginine and sulfonylureas during labelling resulted in a significantly enhanced relative fractional release of newly synthesized (pro-)insulin, although the bulk of secreted hormone appeared to stem from the storage pool also under these conditions. The enhanced fractional release was persistent also during the postlabelling period when the islets had been labelled in the presence of arginine or glibenclamide. On the other hand, a decreased release of newly synthesized (pro-)insulin was observed during the postlabelling period in islets labelled in the presence of tolbutamide. Lysine was without significant effects in both periods. Omission of calcium ions during the postlabelling period inhibited the release of both immunoreactive and radioactive hormone. 2) When amino acids or sulphonylureas were added after prelabelling no signifcant changes were found in the specific radioactivity of released (pro-)insulin or in the fractional release of newly synthesized hormone. Enhanced release of fresh granules from the beta cell might explain the increased fractional release of newly synthesized (pro-) insulin when labelling is carried out in the presence of arginine and sulphonylureas, especially glibenclamide.     

Interacting Effects of Sulfonylureas and Glucose on Cyclic AMP Metabolism and Insulin Release in Pancreatic Islets of the Rat

The effects of tolbutamide and glibenclamide on the metabolism of cyclic AMP were investigated in pancreatic islets of the rat. Changes in cyclic AMP were assessed by measuring [(3)H]cyclic AMP after labeling of the islets with [2-(3)H]adenine. In the presence of a nonstimulatory concentration of glucose (3.3 mM), both sulfonylureas caused a rapid increase in islet [(3)H]cyclic AMP, which declined within 5 (tolbutamide) or 10 min (glibenclamide). In the absence of glucose, the glibenclamide effect was shortened, but the initial (1 min) response of [(3)H]-cyclic AMP was unaffected. Glucose could be substituted with d-glyceraldehyde but not pyruvate for prolongation of the glibenclamide response. The effect of glucose withdrawal on the glibenclamide response was reproduced by the addition of d-mannoheptulose to glucose containing media.The [(3)H]cyclic AMP response to glibenclamide was influenced by prior exposure of the islets to glucose, a 30-min preincubation with 27.7 mM glucose, enhancing the response to the sulfonylurea over a subsequent 5-min stimulation period.Sulfonylureas exerted their effects at low but not at high glucose concentrations, i.e., shifted the glucose dose-response curve to the left both for [(3)H]cyclic AMP accumulation and insulin release. On the other hand, increasing concentrations of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, progressively augmented the effects of the drugs.Omission of Ca(++) from the incubation media inhibited both the glucose and the sulfonylurea [(3)H]-cyclic AMP and insulin responses. Epinephrine (1 muM) partially inhibited the [(3)H]cyclic AMP response to both glucose and sulfonylurea, whereas insulin release was completely abolished.It is concluded that the sulfonylurea effects on islet cyclic AMP are intimately related to those of glucose. It is suggested that sulfonylureas exert a major part of their action by facilitating the effect of glucose on the beta-cell adenylate cyclase; the increased cyclic AMP level, in its turn, enhances the secretion rate of insulin.     

Insulin and Glucagon Secretion by Pancreatic Islets from Nondiabetic and Diabetic Sand Rats (Psammomys obesus)

Abstract. The secretion of insulin and glucagon was investigated in pancreatic islets from diabetic and nondiabetic sand rats of similar age and weight. The metabolic characterization was based on an intraperitoneal glucose tolerance test. Compared to nondiabetic animals diabetic sarid rats had a diminished insulin content in their islets and a decreased insulin secretory response to glucose, glyceraldehyde and theophylline. Diazoxide inhibited insulin release in diabetic as well as in nondiabetic sand rats whereas mannoheptulose was effective only in the nondiabetic rats. There was no significant difference in glucagon content between the two groups. The glucagon secretion by pancreatic islets of diabetic animals was not suppressed by glucose, as in nondiabetic sand rats islets, but by glyceraldehyde. This indicates that the sensitivity to glucose rather than the suppressibility of glucagon release was altered.     

Role of microtubules in the synthesis, conversion, and release of (pro)insulin. A biochemical and radioautographic study in rat islets.

In the pancreatic B cell, microtubules are thought to be involved in the process of insulin release. Their possible participation in the sequence of events leading from the biosynthesis and conversion of proinsulin to the release of newly synthesized insulin was investigated in rat isolated islets exposed to colchicine (0.1 mM). When the islets were preincubated for 30 min with colchicine and [3H]-leucine and, thereafter, incubated for two successive periods of 90 min each, still in the presence of colchicine, the release of preformed insulin was progressively inhibited and that of newly synthesized hormone delayed. When the islets were preincubated for 120 min with colchicine, subsequently pulse-labeled with [3H]leucine, and eventually examined by ultrastructural autoradiography, the export of newly synthesized proinsulin out of the rough endoplasmic reticulum, its transit through the Golgi complex, and its eventual packaging in secretory granules were all retarded. This situation was associated with a delayed conversion of proinsulin to insulin. Under the same experimental conditions, colchicine failed to affect the oxidation of glucose and adenylate charge in the islets. The effect of colchicine upon the release of preformed and newly synthesized insulin was not reproduced by lumicolchicine. It is concluded that colchicine interferes with the system controlling the intracellular transfer of secretory material from site of synthesis to site of release. This interference is likely to be linked to the effect of colchicine on microtubules.     

Enzyme-induced modifications of beta cell function. I. Effect of pronase on insulin secretion

Abstract. The effects upon immunoreactive insulin (IRI) release of beta cell membrane modifications induced by pronase, a mixture of proteolytic enzymes extracted from Streptomyces griseus, have been studied in isolated islets of Langerhans. Pretreatment of the islets for 90 min with 4 μg/ml pronase did not modify their IRI content; it slightly increased the basal rate of IRI release and potentiated the secretory response to glucose, leucine and tolbutamide during incubation. In perifused islets, the rapid phase of IRI secretion in response to glucose was more markedly enhanced than the late phase. After preincubation with 4 μg/ml pronase, glucose-induced IRI release was reversible and abolished in the absence of calcium. Pretreatment for 90 min with 500 μg/ml pronase decreased IRI content of the islets by approximately 25% and provoked a pronounced but transient rise of basal IRI release. This was considered to be a leakage of IRI from damaged beta cells since it persisted in a medium deprived of calcium. The rapid secretory phase in response to glucose was preserved in perifused islets whereas the late phase was markedly reduced. The secretory responses to leucine and tolbutamide were almost completely obliterated. When pretreatment with pronase was carried out in a calcium-depleted medium, basal IRI release from islets preincubated with 500 μg/ml pronase was only slightly increased whereas IRI secretion induced by glucose was inhibited by 40 and 65%, respectively in islets pretreated with 4 and 500 μg/ml pronase. These results indicate that pronase-induced modifications of the beta cell membrane influence IRI secretion in a way which depends on the concentration of the enzyme and the presence of extracellular calcium. They are considered to support the hypothesis that membrane systems are involved in IRI releasing mechanisms.     

Virus-induced alterations in insulin release in hamster islets of Langerhans.

After the inoculation of Golden Syrian hamsters with the TC-83 vaccine strain of Venezuelan encephalitis (VE) virus, a sustained diminution in glucose-stimulated insulin release and glucose intolerance of shorter duration develops. To understand better the mechanism of this defect in insulin release, we examined insulin secretion in response to several test agents in isolated perifused islets from control and 24-d post-VE virus-infected hamsters. 50 islets were used in all perifusion experiments, and data were expressed as total insulin released as well as peak response for each test agent during a 30-min perifusion period from control and VE-infected islets. After perifusion with 20 mM glucose, a 45% diminution of insulin release was noted in VE-infected islets in comparison with control islets, which in turn was similar to in vivo findings. However, following 1-mM tolbutamide stimulation, insulin release was similar in control and VE-infected islets. In separate studies, 1 mM tolbutamide, 10 mM theophilline, 1 mM dibutyryl cyclic (c)AMP, and 1 mM 8-bromo-cAMP resulted in statistically similar insulin-release curves in control and VE-infected islets. Additional experiments assessing [5-3H]glucose use in control and infected islets after 20 min of perifusion with 20 mM glucose revealed virtually identical values (239 +/- 30-control; and 222 +/- 27-VE-infected islets). Morphological and morphometric evaluation of VE-infected islets (21 d following virus inoculation) showed no changes in islet volume density, beta cell density, and beta cell granulation. Thus, VE virus induces a defect in glucose-stimulated insulin release from hamster beta cells that can be corrected by cAMP analogues and does not alter islet glucose use.     

Diabetes Mellitus in Gonadal Dysgenesis: Studies of Insulin and Growth Hormone Secretion

On the basis of results obtained from an oral glucose tolerance test, (OGTT), twenty patients with gonadal dysgenesis were classified as normal (N = 8) and diabetic (N = 12). The two groups of patients were further tested by a rapid intravenous glucose injection, a tolbutamide test, an insulin sensitivity test and an oral amino acid load. Fasting levels of plasma growth hormone (GH) were normal in all subjects but one. Approximately 1/3 of the GH responses during testing periods were abnormal, being either absent during hypoglycaemia or following amino acid ingestion, or paradoxically increased during hyperglycaemia. No correlation was found between the degree of carbohydrate intolerance and the levels of plasma GH. There was no gross alteration of tissue sensitivity to exogenous insulin. The beta-cell response to tolbutamide, amino acid and intravenous glucose were comparable in patients with a normal or a diabetic OGTT. In both groups, the rates of decrease of blood glucose following tolbutamide or intravenous glucose were also similar and within the normal range. During OGTT, the diabetic group had a delayed insulin release and a low insulinogenic index. It is concluded that in gonadal dysgenesis the intolerance to an oral carbohydrate load is frequently associated with, but unrelated to, anomalies of the GH secretion. In diabetic subjects, the process of insulin secretion loses its normal sensitivity to the oral glucose stimulus while remaining unaltered and similiar to that of non-diabetic subjects in response to tolbutamide, amino acid and intravenous glucose.     

Effect of glibenclamide on insulin release at moderate and high blood glucose levels in normal man

Insulin release occurs in two phases; sulphonylurea derivatives may have different potencies in stimulating first- and second-phase insulin release. We studied the effect of glibenclamide on insulin secretion at submaximally and maximally stimulating blood glucose levels with a primed hyperglycaemic glucose clamp. Twelve healthy male subjects, age (mean ± SEM) 22.5 ± 0.5 years, body mass index (BMI) 21.7 ± 0.6 kg m^?2, were studied in a randomized, double-blind study design. Glibenclamide 10 mg or placebo was taken before a 4-h hyperglycaemic clamp (blood glucose 8 mmol L^?1 during the first 2 h and 32 mmol L^?1 during the next 2 h). During hyperglycaemic clamp at 8 mmol L^?1, the areas under the Δinsulin curve (AUC_Δinsulin , mean ± SEM) from 0 to 10 min (first phase) were not different: 1007 ± 235 vs. 1059 ± 261 pmol L^?1 × 10 min (with and without glibenclamide, P = 0.81). However, glibenclamide led to a significantly larger increase in AUC_Δinsulin from 30 to 120 min (second phase): 16 087 ± 4489 vs. 7107 ± 1533 pmol L^?1 × 90 min (with and without glibenclamide respectively, P < 0.03). The same was true for AUC_ΔC-peptide: no difference from 0 to 10 min but a significantly higher AUC_ΔC-peptide from 30 to 120 min on the glibenclamide day (P < 0.01). The M/I ratio (mean glucose infusion rate divided by mean plasma insulin concentration) from 60 to 120 min, a measure of insulin sensitivity, did not change: 0.26 ± 0.05 vs. 0.22 ± 0.03 μmol kg^?1 min^?1 pmol L^?1 (with and without glibenclamide, P = 0.64). During hyperglycaemic clamp at 32 mmol L^?1, the AUC_Δinsulin from 120 to 130 min (first phase) was not different on both study days: 2411 ± 640 vs. 3193 ± 866 pmol L^?1 × 10 min (with and without glibenclamide, P = 0.29). AUC_Δinsulin from 150 to 240 min (second phase) also showed no difference: 59 623 ± 8735 vs. 77389 ± 15161 pmol L^?1 × 90 min (with and without glibenclamide, P = 0.24). AUC_ΔC-peptide from 120 to 130 min and from 150 to 240 min were slightly lower on the glibenclamide study day (both P < 0.04). The M/I ratio from 180 to 240 min did not change: 0.24 ± 0.04 vs. 0.30 ± 0.07 μmol kg^?1 min^?1 pmol L^?1 (with and without glibenclamide, P = 0.25). In conclusion, glibenclamide increases second-phase insulin secretion only at a submaximally stimulating blood glucose level without enhancement of first-phase insulin release and has no additive effect on insulin secretion at maximally stimulating blood glucose levels. Glibenclamide did not change insulin sensitivity in this acute experiment.     

Enzyme-Induced Modifications of Beta Cell Function

Abstract. The effects upon immunoreaetive insulin (IRI) release of beta cell membrane modifications induced by pronase, a mixture of proteolytic enzymes extracted from Streptomyces griseus , have been studied in isolated islets of Langerbans. Pretreatment of the islets for 90 min with 4 μg/ml pronase did not modify their IRI content; it slightly increased the basal rate of IRI release and potentiated the secretory response to glucose, leucine and tolbutamide during incubation. In perifused islets, the rapid phase of IRI secretion in response to glucose was more markedly enhanced than the late phase. After preincubation with 4 μg/ml pronase, glucose-induced IRI release was reversible and abolished in the absence of calcium. Pretreatment for 90 min with 500 μg/ml pronase decreased IRI content of the islets by approximately 25% and provoked a pronounced but transient rise of basal IRI release. This was considered to be a leakage of IRI from damaged beta cells since it persisted in a medium deprived of calcium. The rapid secretory phase in response to glucose was preserved in perifused islets whereas the late phase was markedly reduced. The secretory responses to leucine and tolbutamide were almost completely obliterated. When pretreatment with pronase was carried out in a calcium-depleted medium, basal IRI release from islets preincubated with 500 μg/ml pronase was only slightly increased whereas IRI secretion induced by glucose was inhibited by 40 and 65%, respectively in islets pre treated with 4 and 500 μg/ml pronase. These results indicate that pronase-induced modifications of the beta cell membrane influence IRI secretion in a way which depends on the concentration of the enzyme and the presence of extracellular calcium. They are considered to support the hypothesis that membrane systems are involved in IRI releasing mechanisms.     

Iron Excess, Early Glucose Intolerance and Impaired Insulin Secretion in Idiopathic Haemochromatosis

Abstract. The relationship between iron storage and glucose metabolism was studied in 21 relatives of 4 patients with idiopathic haemochromatosis and in 10 healthy control subjects. In all individuals, plasma iron and iron binding capacity were measured and liver function was assessed. In addition, intravenous and oral glucose tolerance tests (IVGTT, OGTT), as well as tolbutamide (TTT) and insulin tolerance tests (ITT), were performed. Serum insulin (IRI) was measured. Liver biopsies were performed on the 21 relatives only.
In the relatives of patients with haemochromatosis, glucose tolerance was impaired and insulin secretion in response to hyperglycaemia diminished and/or delayed. Glucose intolerance increased with age but did not depend on abnormal liver function or excessive iron storage. Insulin release in response to tolbutamide was normal and insulin tolerance test failed to reveal insulin resistance.
The results suggest that:
1. There is an early glucose intolerance in healthy relatives of patients suffering from idiopathic haemochromatosis.
2. The glucose intolerance seems unrelated to measurable anomalies in iron metabolism.
3. The delayed insulin secretion in response to glucose resembles that observed in common maturity-onset diabetes mellitus.
4. The results obtained are compatible with the hypothesis that haemochromatosis and diabetes mellitus might be two distinct but genetically linked entities.     

Gosha-jinki-gan (a Herbal Complex) Corrects Abnormal Insulin Signaling

Previous studies have shown that the traditional herbal complexGosha-jinki-gan (GJG) improves diabetic neuropathy and insulinresistance. The present study was undertaken to elucidate themolecular mechanisms related with the long-term effects of GJGadministration on insulin action in vivo and the early stepsof insulin signaling in skeletal muscle in streptozotocin (STZ)diabetes. Rats were randomized into five subgroups: (1) salinetreated control, (2) GJG treated control, (3) 2-unit insulin+ saline treated diabetic, (4) saline + GJG treated diabeticand (5) 2-unit insulin + GJG treated diabetic groups. Afterseven days of treatment, euglycemic clamp experiment at an insulininfusion rate of 6 mU/kg/min was performed in overnight fastedrats. Despite the 2-unit insulin treatment, the metabolic clearancerates of glucose (MCR, ml/kg/min) in diabetic rats were significantlylower compared with the controls (11.4 ± 1.0 vs 44.1± 1.5; P < 0.001), and were significantly improvedby insulin combined with GJG or GJG alone (26 ± 3.2 and24.6 ± 2.2, P < 0.01, respectively). The increasedinsulin receptor (IR)-ß protein content in skeletalmuscle of diabetic rats was not affected by insulin combinedwith GJG administration. However, the decreased insulin receptorsubstrate-1 (IRS-1) protein content was significantly improvedby treatment with GJG. Additionally, the increased tyrosinephosphorylation levels of IR-ß and IRS-1 were significantlyinhibited in insulin combined with GJG treated diabetes. Thepresent results suggest that the improvement of the impairedinsulin sensitivity in STZ-diabetic rats by administration ofGJG may be due, at least in part, to correction in the abnormalearly steps of insulin signaling in skeletal muscle.     

Pulsatile insulin release from pancreatic islets with nonoscillatory elevation of cytoplasmic Ca2+.

The relationship between insulin release and cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in isolated pancreatic islets from ob/ob mice. Although [Ca2+]i was low and stable in the presence of 3 mM glucose, basal insulin release exhibited low amplitude pulsatility, with a frequency of 0.32 +/- 0.04 min-1. Depolarization by raising K+ from 5.9 to 30.9 mM or by the addition of 1 mM tolbutamide caused a pronounced initial insulin pulse followed by declining pulses, but there was no change in frequency. This decline in amplitude of the insulin pulses was prevented in similar experiments performed in the presence of 11 mM glucose. Corresponding measurements of [Ca2+]i in islets exposed to tolbutamide or the high K+ concentration revealed stable elevations without oscillations. Although the [Ca2+]i level is an important determinant for the rate of secretion, the results indicate that pulsatile insulin release does not always depend on [Ca2+]i oscillations. It is suggested that cyclic generation of ATP may fuel pulsatile release under conditions when [Ca2+]i remains stable.     

Comparison of the effect of GIP and GLP-1 (7–36amide) on insulin release from rat pancreatic islets

After ingestion of glucose both GIP (gastric inhibitory polypeptide, glucose-dependent insulinotropic polypeptide) and GLP-1(7-36amide) (glucagon-like polypeptide-1, 7-36amide) may play a physiological role in augmenting insulin release. Their insulinotropic effect was compared in isolated rat islets after 24-h maintenance in tissue culture (11 mmol l-1 glucose). Ten islets per vial were then incubated in Krebs-Ringer-Hepes buffer for 30 min; insulin was measured radioimmunologically. Both hormones were always compared in the same experiment. At 16.7 mmol l-1 glucose both GIP and GLP-1(7-36amide) 2 x 10(-10) mol l-1 significantly increased insulin release; 10(-10) mol l-1 of either hormone had no significant effect. The response at 10(-9) and 10(-8) mol l-1 was similar for both; at 4 x 10(-10) mol l-1 GLP-1(7-36amide), however, was clearly more effective than GIP. At low glucose (2.8 or 5.0 mol l-1) no significant differences were found. A concentration of 10(-8) mol l-1 of both hormones was slightly stimulatory. At 8.3 mmol l-1 glucose, 10(-9) mol l-1 GLP-1(7-36amide) was 60% more effective than GIP (4.8 +/- 0.4 vs. 3.0 +/- 0.4, n = 13, P less than 0.005), the response to 10(-8) mol l-1 was similar. These data show comparable effects of high concentrations of GIP and GLP-1(7-36amide) on glucose-induced insulin release; at presumably physiological concentrations, however, GLP-1(7-36amide) was clearly more effective. The combination of the two peptides was not more than additive, suggesting that they act via the same final mechanism.     

Synthesis and release of proinsulin and insulin by isolated rat islets of Langerhans

Isolated rat islets of Langerhans were incubated for 60, 120, and 180 min and the incorporation of leucine-(3)H into proinsulin and insulin moieties was followed. Synthesis and release of these hormones could be followed by separate extractions of islets and incubation media.RELEASE OF NEWLY SYNTHESIZED PROINSULIN AND INSULIN OCCURRED UNDER THE FOLLOWING CONDITIONS: (a) incubation for greater than 60 min; (b) glucose concentrations above 5.3 mmoles/liter; (c) incubation with 5 mM dibutyryl cyclic AMP or theophylline in 5.3 mM glucose (potentiated by 16 mM glucose); and (d) incubation with 5 mM tolbutamide and 16 mM glucose.Synthesis of proinsulin and insulin was enhanced by time of incubation, high glucose concentrations, by dibutyryl cyclic AMP or theophylline, and by tolbutamide only at 16 mM glucose. Synthesis was totally inhibited by tolbutamide at 5.3 mM glucose.     

Effects of Burn Injury on Insulin Secretion and on Sensitivity to Insulin in the Rat in vivo

Abstract. Both insulin resistance and impairment of insulin secretion are know to occur in man after injury. The relative importance of these effects was studied in rats 2 h after a non-lethal 20 % dorsal scald. No impairment of insulin secretion was found after this injury. Concentrations of both blood glucose and plasma insulin were elevated in scalded rats. Scalded rats responded to intravenous glucose injection (1. 0 g/kg) with a further rise in plasma insulin concentration, which remained normal for the prevailing blood glucose concentration. However, marked impairment of glucose tolerance was observed, indicating the presence of insulin resistance. After intravenous insulin injection (1. 0 U/kg) the initial rate coefficient for fall of blood glucose concentration was significantly lower (p < 0–02) in scalded (mean 3. 9 % min. ^-1) than in control rats (mean 6–3 % min. ^-1). The minimum in blood glucose concentration after insulin injection was reached at 10 min. in control rats, but not until 60 min. after injection in scalded rats. This difference was due to a delay in compensation for the hypoglycaemia in the scalded rats, since the rate of disappearance of insulin measured by injection of a tracer of 25i-labelled bovine insulin was not decreased after this injury. It was concluded that the impairment of glucose utilization in scalded rats (Heath and Corney, 1973) is due to decreased sensitivity to insulin rather than to suppression of insulin release.     

In Vitro Studies of the Rate of Proinsulin and Insulin Turnover in Seven Human Insulinomas

Summary. Human insulinoma tissue excised from seven patients was incubated with glucose (3.0 mg/ml) and ³H-leucine (100 μCi/ml) for a 15 min. pulse period. One third of the tissue was homogenized immediately in 0.3 M sucrose (pH 6.0), one third was homogenized after a 20 min. chase (with L-leueine 37S μg/ml) and the remaining third after an 80 min. chase. Subcellular fractions (cell debris, mitochondria, secretory granules, microsomes and S-100) were prepared by centrifugation. Their identity was confirmed by ultrastructural examination. Since only tumours revealing typical β-granules were investigated the secretory granule fractions contained the same types of granules as found in the normal human B-cell.—The S-100 contained approximately 40% of the immuno-reactive insulin (IRI). The secretory granule samples from five tumours were fractionated by sucrose gradient ultracentrifugation. At the three time intervals examined, a peak of radioactivity and IRI was located at 1.60 M sucrose in three of the tumours. The different granule types were not separable in the sucrose gradients. Endogenous proinsulin-like components (proinsulin) and insulin were released throughout the incubation. During incubation of tumour tissue the ratio of IRI release to content was significantly higher than that from isolated human pancreatic islets. Newly synthesized proinsulin was detected in the medium after the 15 min. pulse in five of the tumours; radioactive proinsulin and insulin were present in all the media after the 20 and 80 min. chase periods. Islets isolated from the pancreatic tissue of two insulinoma patients showed a much slower proinsulin and insulin turnover.—These data support the conjecture that human insulinomas have a more rapid turnover of proinsulin and insulin than normal pancreatic islet tissue, apparently as the consequence of defective IRI storage and release mechanisms.     

Insulin sensitivity (Si) assessment in lean and overweight subjects using two different protocols and updated software

Abstract

Background. The standard frequently-sampled intravenous glucose tolerance test (FSIVGTT) is an alternative procedure to the clamp technique for estimating the insulin sensitivity (Si) parameter. The goal of this study was to compare Si in lean and overweight individuals in addition to assessing intra-individual reproducibility using two different protocols and updated software. Methods. FSIVGTT was carried out in 14 lean (BMI ≤ 25 kg/m²) and 14 overweight (BMI>25 kg/m²) subjects using two different protocols; full (29 samples) and short (12 samples). For reproducibility assessment four normal subjects (triplicate on three and twice on one) were recruited to undergo the same procedure at 1-week intervals. Data analysis was performed using COMAL and Minmod Millennium software. Results. Mean Si (10^?4min^?1[pmol/l]^?1) values were significantly different between lean and overweight subjects (p < 0.001) but not between the two protocols using both software packages. For the full and short protocols, Si values were more closely related in lean versus overweight subjects using either COMAL (r = 0.98, p < 0.001), (r = 0.89, p < 0.001) or Minmod Millennium (r = 0.99, p < 0.001), (r = 0.85, p < 0.001) software respectively. The intra-individual reproducibility (%CV) of Si (COMAL) in full versus short protocol was 18.3 ± 11.1% and 13.7 ± 1.9% respectively. Reproducibility values for Si (Minmod Millenium) in full versus short protocols were 14.3 ± 3.8 and 14.9 ± 1.9% respectively. Conclusions. Si can be assessed accurately by a short protocol FSIVGTT in normal individuals. The short protocol may give less acceptable results for insulin sensitivity in individuals who have normal glucose tolerance but high BMI.     

Somatostatin- and Epinephrine-Induced Modifications of 45Ca++ Fluxes and Insulin Release in Rat Pancreatic Islets Maintained in Tissue Culture

The effects of somatostatin and epinephrine have been studied with regard to glucose-induced insulin release and (45)Ca(++) uptake by rat pancreatic islets after 2 days in tissue culture and with regard to (45)Ca(++) efflux from islets loaded with the radio-isotope during the 2 days of culture. (45)Ca(++) uptake, measured simultaneously with insulin release, was linear with time for 5 min. (45)Ca(++) efflux and insulin release were also measured simultaneously from perifused islets.Glucose (16.7 mM) markedly stimulated insulin release and (45)Ca(++) uptake. Somatostatin inhibited the stimulation of insulin release by glucose in a concentration-related manner (1-1,000 ng/ml) but was without effect on the glucose-induced stimulation of (45)Ca(++) uptake. Similarly, under perifusion conditions, both phases of insulin release were inhibited by somatostatin while no effect was observed on the pattern of (45)Ca(++) efflux after glucose.Epinephrine, in contrast to somatostatin, caused a concentration-dependent inhibition of the stimulation of both insulin release and (45)Ca(++) uptake by glucose. Both phases of insulin release were inhibited by epinephrine and marked inhibition could be observed with no change in the characteristic glucose-evoked pattern of (45)Ca(++) efflux (e.g., with 10 nM epinephrine). The inhibitory effect of epinephrine on (45)Ca(++) uptake and insulin release appeared to be mediated via an alpha-adrenergic mechanism, since is was abolished in the presence of phentolamine.Somatostatin inhibits insulin release without any detectable effect upon the handling of calcium by the islets. In contrast, inhibition of insulin release by epinephrine is accompanied by a partial inhibition of glucose-induced Ca(++) uptake.     

Inhibition of Na/Ca exchange stimulates insulin release from isolated rat pancreatic islets

Summary— Na/Ca exchange was recently shown to regulate cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic B-cell. The aim of the present study was to provide direct evidence that inhibition of the activity of the exchange may also increase insulin release. In the presence of extracellular Na+, caffeine stimulated ⁴⁵Ca outflow but did not increase insulin release from islets perifused in the presence of 2.8 mM glucose. By contrast, in the absence of extracellular Na+, caffeine almost failed to increase ⁴⁵Ca outflow and reversibly stimulated insulin release despite the fact that the absence of extracellular Na+ per se reduced basal insulin release. Similar findings were observed in islets perifused at a higher glucose concentration (8.3 mM) except that, in the presence of extracellular Na+, caffeine more markedly increased ⁴⁵Ca outflow and stimulated insulin release. Our data provide direct evidence that inhibition of Na/Ca exchange with resulting blockade of Ca²⁺ outflow may increase insulin release from the pancreatic B-cell under suitable experimental conditions.     

Contents and secretion of glucagon and insulin in rat pancreatic islets from the viewpoint of their localization in pancreas

Regional difference in secretion of glucagon and insulin from the rat pancreatic islets and their contents in pancreatic tissue and islets were studied. The glucagon content in the normal rat pancreas was the highest at the splenic part followed in order by the gastric, choledochal and duodenal parts. The effect of alloxan on the glucagon content was stronger in the dorsal lobe (combined gastric and splenic parts) than in the ventral lobe (combined duodenal and choledochal parts), and more increase of glucagon was found. The size of pancreatic islets was similar between the dorsal and the ventral lobe. The glucagon content in the islets was significantly higher in the dorsal lobe than in the ventral lobe, but the insulin content in the islets was similar in both lobes. The release of insulin from cultured pancreatic islets from the dorsal lobe was similar to that from the ventral lobe, but the release of glucagon was significantly high from the cultured islets of the former in the presence of glucose or arginine compared with that from the latter. Also in isolated pancreatic islets the release of glucagon was significantly more marked in the islets from the dorsal lobe by arginine administration. These findings show that the islets from the dorsal lobe secrete and contain more glucagon than those from the ventral lobe in contrast to there being a similar amount of release of insulin between them.